Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

RELATED APPLICATIONS

The present application claims priority to provisional application U.S. Ser. No. 60/232,633, filed Sep. 14, 2000.

FIELD OF THE INVENTION

The present invention is in the field of kinase proteins that are related to the protein kinase C subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION Protein Kinases

Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for controlling activities of eukaryotic cells. It is estimated that more than 1000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate, which drives activation, is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc), cell cycle checkpoints, and environmental or nutritional stresses and is roughly analogous to turning on a molecular switch. When the switch goes on, the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.

The kinases comprise the largest known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate threonine and tyrosine residues. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain, which contains subdomains I-IV, generally folds into a two-lobed structure, which binds and orients the ATP (or GTP) donor molecule. The larger C terminal lobe, which contains subdomains VI A-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.

The kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. Each of the 11 subdomains contains specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Books, Vol I.:7-20 Academic Press, San Diego, Calif.).

The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic-ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) are important members of the STK family. Cyclic-AMP is an intracellular mediator of hormone action in all prokaryotic and animal cells that have been studied. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction. PKA is found in all animal cells and is thought to account for the effects of cyclic-AMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York, N.Y., pp. 416-431, 1887).

Calcium-calmodulin (CaM) dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases. CaM-kinases are involved in regulation of smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal 14:3679-86). CaM II kinase also phosphorylates synapsin at different sites, and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. Many of the CaM kinases are activated by phosphorylation in addition to binding to CaM. The kinase may autophosphorylate itself, or be phosphorylated by another kinase as part of a “kinase cascade”.

Another ligand-activated protein kinase is 5′-AMP-activated protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81). Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP. AMPK is a heterotrimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit. Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected. This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.

The mitogen-activated protein kinases (MAP) are also members of the STK family. MAP kinases also regulate intracellular signaling pathways. They mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature 365:781-783). MAP kinase signaling pathways are present in mammalian cells as well as in yeast. The extracellular stimuli that activate mammalian pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).

PRK (proliferation-related kinase) is a serum/cytokine inducible STK that is involved in regulation of the cell cycle and cell proliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem. 271:19402-8). PRK is related to the polo (derived from humans polo gene) family of STKs implicated in cell division. PRK is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation. Altered MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.

The cyclin-dependent protein kinases (CDKs) are another group of STKs that control the progression of cells through the cell cycle. Cyclins are small regulatory proteins that act by binding to and activating CDKs that then trigger various phases of the cell cycle by phosphorylating and activating selected proteins involved in the mitotic process. CDKs are unique in that they require multiple inputs to become activated. In addition to the binding of cyclin, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue.

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosine residues on their target proteins and may be divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane protein-tyrosine kinases are receptors for most growth factors. Binding of growth factor to the receptor activates the transfer of a phosphate group from ATP to selected tyrosine side chains of the receptor and other specific proteins. Growth factors (GF) associated with receptor PTKs include; epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.

Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Such receptors that function through non-receptor PTKs include those for cytokines, hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes.

Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells where their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Carbonneau H and Tonks N K (1992) Annu. Rev. Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer.

Protein Kinase C

Protein kinase C (PKC) proteins are members of the STK family. Protein kinase D (PKD) proteins bind phorbol esters and diacylglycerol and are closely related to PKCs (Valverde et al., Proc Natl Acad Sci USA Aug. 30, 1994;91(18):8572-6).

Protein kinase C plays a key role in modulating cellular responses in a wide variety of extracellular receptor-mediated signal transduction pathways, and in regulating cellular differentiation and proliferation in a wide variety of cells.

Protein kinase C genes/proteins may play an important role in many cancers, and therefore may be useful for drug development and for screening for, diagnosing, preventing, and/or treating a variety of cancers. For example, tumor-specific deletions have been identified within the gene for alpha-type protein kinase C in a melanoma cell line (Linnenbach et al., Proc Natl Acad Sci USA 1988 January; 85(1):74-8). Elevated expression levels of PKCs have been observed in certain tumor cell lines and it has been suggested that PKCs play an important role in signal transduction pathways related to growth control (Johannes et al., J Biol Chem Feb. 25, 1994; 269(8):6140-8).

For a further review of PKCs, see Owczarek et al., Cytogenet. Cell Genet. 89: 240-241, 2000 and Hayashi et al., Biochim Biophys Acta May 6, 1999; 1450(1):99-106.

Kinase proteins, particularly members of the protein kinase C subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of kinase proteins. The present invention advances the state of the art by providing previously unidentified human kinase proteins that have homology to members of the protein kinase C subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human kinase peptides and proteins that are related to the protein kinase C subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate kinase activity in cells and tissues that express the kinase. Experimental data as provided in FIG. I indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the kinase protein of the present invention. (SEQ ID NO: 1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas.

FIG. 2 provides the predicted amino acid sequence of the kinase of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the kinase protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As indicated in FIG. 3, SNPs were identified at 44 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION GENERAL DESCRIPTION

The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a kinase protein or part of a kinase protein and are related to the protein kinase C subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human kinase peptides and proteins that are related to the protein kinase C subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these kinase peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the kinase of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known kinase proteins of the protein kinase C subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known protein kinase C family or subfamily of kinase proteins.

SPECIFIC EMBODIMENTS Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the kinase family of proteins and are related to the protein kinase C subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the kinase peptides of the present invention, kinase peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the kinase peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the kinase peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated kinase peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. For example, a nucleic acid molecule encoding the kinase peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the kinase peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The kinase peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a kinase peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the kinase peptide. “Operatively linked” indicates that the kinase peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the kinase peptide.

In some uses, the fusion protein does not affect the activity of the kinase peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant kinase peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the kinase peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the kinase peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the kinase peptides of the present invention as well as being encoded by the same genetic locus as the kinase peptide provided herein. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 19 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

Allelic variants of a kinase peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by the same genetic locus as the kinase peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 19 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase proteins of the present invention. SNPs were identified at 44 different nucleotide positions, including a non-synonymous coding SNP at position 42934. The change in the amino acid sequence that this SNP causes is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements.

Paralogs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

Non-naturally occurring variants of the kinase peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the kinase peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a kinase peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant kinase peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as kinase activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the kinase peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a kinase peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the kinase peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the kinase peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in kinase peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N. Y Acad. Sci. 663:48-62 (1992)).

Accordingly, the kinase peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature kinase peptide is fused with another compound, such as a compound to increase the half-life of the kinase peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature kinase peptide, such as a leader or secretory sequence or a sequence for purification of the mature kinase peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a kinase-effector protein interaction or kinase-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, kinases isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the kinase. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Specifically, a virtual northern blot shows expression in lung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissue screening panels indicate expression in bone marrow, brain (including fetal), colon, heart (including fetal), kidney, lung (including fetal), fetal liver, uterus, and pancreas. A large percentage of pharmaceutical agents are being developed that modulate the activity of kinase proteins, particularly members of the protein kinase C subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to kinases that are related to members of the protein kinase C subfamily. Such assays involve any of the known kinase functions or activities or properties useful for diagnosis and treatment of kinase-related conditions that are specific for the subfamily of kinases that the one of the present invention belongs to, particularly in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Specifically, a virtual northern blot shows expression in lung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissue screening panels indicate expression in bone marrow, brain (including fetal), colon, heart (including fetal), kidney, lung (including fetal), fetal liver, uterus, and pancreas.

The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the kinase, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the kinase protein.

The polypeptides can be used to identify compounds that modulate kinase activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the kinase. Both the kinases of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the kinase. These compounds can be further screened against a functional kinase to determine the effect of the compound on the kinase activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the kinase to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the kinase protein and a molecule that normally interacts with the kinase protein, e.g. a substrate or a component of the signal pathway that the kinase protein normally interacts (for example, another kinase). Such assays typically include the steps of combining the kinase protein with a candidate compound under conditions that allow the kinase protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the kinase protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant kinases or appropriate fragments containing mutations that affect kinase function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) kinase activity. The assays typically involve an assay of events in the signal transduction pathway that indicate kinase activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the kinase protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the kinase can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the kinase can be assayed. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Specifically, a virtual northern blot shows expression in lung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissue screening panels indicate expression in bone marrow, brain (including fetal), colon, heart (including fetal), kidney, lung (including fetal), fetal liver, uterus, and pancreas.

Binding and/or activating compounds can also be screened by using chimeric kinase proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native kinase. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the kinase is derived.

The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the kinase (e.g. binding partners and/or ligands). Thus, a compound is exposed to a kinase polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble kinase polypeptide is also added to the mixture. If the test compound interacts with the soluble kinase polypeptide, it decreases the amount of complex formed or activity from the kinase target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the kinase. Thus, the soluble polypeptide that competes with the target kinase region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the kinase protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of kinase-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a kinase-binding protein and a candidate compound are incubated in the kinase protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the kinase protein target molecule, or which are reactive with kinase protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the kinases of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of kinase protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the kinase pathway, by treating cells or tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. These methods of treatment include the steps of administering a modulator of kinase activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the kinase proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the kinase and are involved in kinase activity. Such kinase-binding proteins are also likely to be involved in the propagation of signals by the kinase proteins or kinase targets as, for example, downstream elements of a kinase-mediated signaling pathway. Alternatively, such kinase-binding proteins are likely to be kinase inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a kinase protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a kinase-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the kinase protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a kinase-modulating agent, an antisense kinase nucleic acid molecule, a kinase-specific antibody, or a kinase-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

The kinase proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. The method involves contacting a biological sample with a compound capable of interacting with the kinase protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered kinase activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the kinase protein in which one or more of the kinase functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and kinase activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Accordingly, methods for treatment include the use of the kinase protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments of the kinase proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or kinase/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidinibiotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Specifically, a virtual northern blot shows expression in lung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissue screening panels indicate expression in bone marrow, brain (including fetal), colon, heart (including fetal), kidney, lung (including fetal), fetal liver, uterus, and pancreas. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the kinase peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid molecules that encode a kinase peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the kinase peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the kinase peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the kinase proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 19 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase proteins of the present invention. SNPs were identified at 44 different nucleotide positions, including a non-synonymous coding SNP at position 42934. The change in the amino acid sequence that this SNP causes is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2×SSC, 0. 1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As indicated in FIG. 3, SNPs, were identified at 44 different nucleotide positions.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 19 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymnes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Specifically, a virtual northern blot shows expression in lung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissue screening panels indicate expression in bone marrow, brain (including fetal), colon, heart (including fetal), kidney, lung (including fetal), fetal liver, uterus, and pancreas. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in kinase protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a kinase protein, such as by measuring a level of a kinase-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a kinase gene has been mutated. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Specifically, a virtual northern blot shows expression in lung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissue screening panels indicate expression in bone marrow, brain (including fetal), colon, heart (including fetal), kidney, lung (including fetal), fetal liver, uterus, and pancreas.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate kinase nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the kinase gene, particularly biological and pathological processes that are mediated by the kinase in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. The method typically includes assaying the ability of the compound to modulate the expression of the kinase nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired kinase nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the kinase nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for kinase nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the kinase protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of kinase gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of kinase mRNA in the presence of the candidate compound is compared to the level of expression of kinase mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate kinase nucleic acid expression in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Specifically, a virtual northern blot shows expression in lung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissue screening panels indicate expression in bone marrow, brain (including fetal), colon, heart (including fetal), kidney, lung (including fetal), fetal liver, uterus, and pancreas. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the kinase nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. I indicates expression in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the kinase gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in kinase nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in kinase genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the kinase gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the kinase gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a kinase protein.

Individuals carrying mutations in the kinase gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase proteins of the present invention. SNPs were identified at 44 different nucleotide positions, including a non-synonymous coding SNP at position 42934. The change in the amino acid sequence that this SNP causes is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 19 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364(1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a kinase gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method. Furthermore, sequence differences between a mutant kinase gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the kinase gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase proteins of the present invention. SNPs were identified at 44 different nucleotide positions, including a non-synonymous coding SNP at position 42934. The change in the amino acid sequence that this SNP causes is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control kinase gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of kinase protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into kinase protein.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of kinase nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired kinase nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the kinase protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in kinase gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired kinase protein to treat the individual.

The invention also encompasses kits for detecting the presence of a kinase nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that kinase proteins of the present invention are expressed in humans in the lung (including fetal and carcinoid lung tissue), lymph (including mantle cell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain (including fetal), heart (including fetal), fetal liver, uterus, and pancreas. Specifically, a virtual northern blot shows expression in lung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissue screening panels indicate expression in bone marrow, brain (including fetal), colon, heart (including fetal), kidney, lung (including fetal), fetal liver, uterus, and pancreas. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting kinase nucleic acid in a biological sample; means for determining the amount of kinase nucleic acid in the sample; and means for comparing the amount of kinase nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect kinase protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS: 1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/25 1116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the kinase proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the kinase gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase proteins of the present invention. SNPs were identified at 44 different nucleotide positions, including a non-synonymous coding SNP at position 42934. The change in the amino acid sequence that this SNP causes is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. SNPs outside the ORF and in introns may affect control/regulatory elements.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified kinase gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSecl (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufinan et al., EMBO J. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as kinases, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with kinases, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a kinase protein or peptide that can be further purified to produce desired amounts of kinase protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving the kinase protein or kinase protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native kinase protein is useful for assaying compounds that stimulate or inhibit kinase protein function.

Host cells are also useful for identifying kinase protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant kinase protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native kinase protein.

Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a kinase protein and identifying and evaluating modulators of kinase protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the kinase protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the kinase protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, kinase protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo kinase protein function, including substrate interaction, the effect of specific mutant kinase proteins on kinase protein function and substrate interaction, and the effect of chimeric kinase proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more kinase protein functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

4 1 2637 DNA Homo sapiens 1 atggccaccg ccccctctta tcccgccggg ctccctggct ctcccgggcc ggggtctcct 60 ccgccccccg gcggcctaga gctgcagtcg ccgccaccgc tactgcccca gatcccggcc 120 ccgggttccg gggtctcctt tcacatccag atcgggctga cccgcgagtt cgtgctgttg 180 cccgccgcct ccgagctggc tcatgtgaag cagctggcct gttccatcgt ggaccagaag 240 ttccctgagt gtggcttcta cggcctttac gacaagatcc tgcttttcaa acatgacccc 300 acgtcggcca acctcctgca gctggtgcgc tcgtccggag acatccagga gggcgacctg 360 gtggaggtgg tgctgtcggc ctcggccacc ttcgaggact tccagatccg cccgcacgcc 420 ctcacggtgc actcctatcg ggcgcctgcc ttctgtgatc actgcgggga gatgctcttc 480 ggcctagtgc gccagggcct caagtgcgat ggctgcgggc tgaactacca caagcgctgt 540 gccttcagca tccccaacaa ctgtagtggg gcccgcaaac ggcgcctgtc atccacgtct 600 ctggccagtg gccactcggt gcgcctcggc acctccgagt ccctgccctg cacggctgaa 660 gagctgagcc gtagcaccac cgaactcctg cctcgccgtc ccccgtcatc ctcttcctcc 720 tcttctgcct catcgtatac gggccgcccc attgagctgg acaagatgct gctctccaag 780 gtcaaggtgc cgcacacctt cctcatccac agctatacac ggcccaccgt ttgccaggct 840 tgcaagaaac tcctcaaggg cctcttccgg cagggcctgc aatgcaaaga ctgcaagttt 900 aactgtcaca aacgctgcgc cacccgcgtc cctaatgact gcctggggga ggcccttatc 960 aatggagatg tgccgatgga ggaggccacc gatttcagcg aggctgacaa gagcgccctc 1020 atggatgagt cagaggactc cggtgtcatc cctggctccc actcagagaa tgcgctccac 1080 gccagtgagg aggaggaagg cgagggaggc aaggcccaga gctccctggg gtacatcccc 1140 ctaatgaggg tggtgcaatc ggtgcgacac acgacgcgga aatccagcac cacgctgcgg 1200 gagggttggg tggttcatta cagcaacaag gacacgctga gaaagcggca ctattggcgc 1260 ctggactgca agtgtatcac gctcttccag aacaacacga ccaacagata ctataaggaa 1320 attccgctgt cagaaatcct cacggtggag tccgcccaga acttcagcct tgtgccgccg 1380 ggcaccaacc cacactgctt tgagatcgtc actgccaatg ccacctactt cgtgggcgag 1440 atgcctggcg ggactccggg tgggccaagt gggcaggggg ctgaggccgc ccggggctgg 1500 gagacagcca tccgccaggc cctgatgccc gtcatccttc aggacgcacc cagcgcccca 1560 ggccacgcgc cccacagaca agcttctctg agcatctctg tgtccaacag tcagatccaa 1620 gagaatgtgg acattgccac tgtctaccag atcttccctg acgaagtgct gggctcaggg 1680 cagtttggag tggtctatgg aggaaaacac cggaagacag gccgggacgt ggcagttaag 1740 gtcattgaca aactgcgctt ccctaccaag caggagagcc agctccggaa tgaagtggcc 1800 attctgcaga gcctgcggca tcccgggatc gtgaacctgg agtgcatgtt cgagacgcct 1860 gagaaagtgt ttgtggtgat ggagaagctg catggggaca tgttggagat gatcctgtcc 1920 agtgagaagg gccggctgcc tgagcgcctc accaagttcc tcatcaccca gatcctggtg 1980 gctttgagac accttcactt caagaacatt gtccactgtg acttgaaacc agaaaacgtg 2040 ttgctggcat cagcagaccc atttcctcag gtgaagctgt gtgactttgg ctttgctcgc 2100 atcatcggcg agaagtcgtt ccgccgctca gtggtgggca cgccggccta cctggcaccc 2160 gaggtgctgc tcaaccaggg ctacaaccgc tcgctggaca tgtggtcagt gggcgtgatc 2220 atgtacgtca gcctcagcgg caccttccct ttcaacgagg atgaggacat caatgaccag 2280 atccagaacg ccgccttcat gtacccggcc agcccctgga gccacatctc agctggagcc 2340 attgacctca tcaacaacct gctgcaggtg aagatgcgca aacgctacag cgtggacaaa 2400 tctctcagcc acccctggtt acaggagtac cagacgtggc tggacctccg agagctggag 2460 gggaagatgg gagagcgata catcacgcat gagagtgacg acgcgcgctg ggagcagttt 2520 gcagcagagc atccgctgcc tgggtctggg ctgcccacgg acagggatct cggtggggcc 2580 tgtccaccac aggaccacga catgcagggg ctggcggagc gcatcagtgt tctctga 2637 2 878 PRT Homo sapiens 2 Met Ala Thr Ala Pro Ser Tyr Pro Ala Gly Leu Pro Gly Ser Pro Gly 1 5 10 15 Pro Gly Ser Pro Pro Pro Pro Gly Gly Leu Glu Leu Gln Ser Pro Pro 20 25 30 Pro Leu Leu Pro Gln Ile Pro Ala Pro Gly Ser Gly Val Ser Phe His 35 40 45 Ile Gln Ile Gly Leu Thr Arg Glu Phe Val Leu Leu Pro Ala Ala Ser 50 55 60 Glu Leu Ala His Val Lys Gln Leu Ala Cys Ser Ile Val Asp Gln Lys 65 70 75 80 Phe Pro Glu Cys Gly Phe Tyr Gly Leu Tyr Asp Lys Ile Leu Leu Phe 85 90 95 Lys His Asp Pro Thr Ser Ala Asn Leu Leu Gln Leu Val Arg Ser Ser 100 105 110 Gly Asp Ile Gln Glu Gly Asp Leu Val Glu Val Val Leu Ser Ala Ser 115 120 125 Ala Thr Phe Glu Asp Phe Gln Ile Arg Pro His Ala Leu Thr Val His 130 135 140 Ser Tyr Arg Ala Pro Ala Phe Cys Asp His Cys Gly Glu Met Leu Phe 145 150 155 160 Gly Leu Val Arg Gln Gly Leu Lys Cys Asp Gly Cys Gly Leu Asn Tyr 165 170 175 His Lys Arg Cys Ala Phe Ser Ile Pro Asn Asn Cys Ser Gly Ala Arg 180 185 190 Lys Arg Arg Leu Ser Ser Thr Ser Leu Ala Ser Gly His Ser Val Arg 195 200 205 Leu Gly Thr Ser Glu Ser Leu Pro Cys Thr Ala Glu Glu Leu Ser Arg 210 215 220 Ser Thr Thr Glu Leu Leu Pro Arg Arg Pro Pro Ser Ser Ser Ser Ser 225 230 235 240 Ser Ser Ala Ser Ser Tyr Thr Gly Arg Pro Ile Glu Leu Asp Lys Met 245 250 255 Leu Leu Ser Lys Val Lys Val Pro His Thr Phe Leu Ile His Ser Tyr 260 265 270 Thr Arg Pro Thr Val Cys Gln Ala Cys Lys Lys Leu Leu Lys Gly Leu 275 280 285 Phe Arg Gln Gly Leu Gln Cys Lys Asp Cys Lys Phe Asn Cys His Lys 290 295 300 Arg Cys Ala Thr Arg Val Pro Asn Asp Cys Leu Gly Glu Ala Leu Ile 305 310 315 320 Asn Gly Asp Val Pro Met Glu Glu Ala Thr Asp Phe Ser Glu Ala Asp 325 330 335 Lys Ser Ala Leu Met Asp Glu Ser Glu Asp Ser Gly Val Ile Pro Gly 340 345 350 Ser His Ser Glu Asn Ala Leu His Ala Ser Glu Glu Glu Glu Gly Glu 355 360 365 Gly Gly Lys Ala Gln Ser Ser Leu Gly Tyr Ile Pro Leu Met Arg Val 370 375 380 Val Gln Ser Val Arg His Thr Thr Arg Lys Ser Ser Thr Thr Leu Arg 385 390 395 400 Glu Gly Trp Val Val His Tyr Ser Asn Lys Asp Thr Leu Arg Lys Arg 405 410 415 His Tyr Trp Arg Leu Asp Cys Lys Cys Ile Thr Leu Phe Gln Asn Asn 420 425 430 Thr Thr Asn Arg Tyr Tyr Lys Glu Ile Pro Leu Ser Glu Ile Leu Thr 435 440 445 Val Glu Ser Ala Gln Asn Phe Ser Leu Val Pro Pro Gly Thr Asn Pro 450 455 460 His Cys Phe Glu Ile Val Thr Ala Asn Ala Thr Tyr Phe Val Gly Glu 465 470 475 480 Met Pro Gly Gly Thr Pro Gly Gly Pro Ser Gly Gln Gly Ala Glu Ala 485 490 495 Ala Arg Gly Trp Glu Thr Ala Ile Arg Gln Ala Leu Met Pro Val Ile 500 505 510 Leu Gln Asp Ala Pro Ser Ala Pro Gly His Ala Pro His Arg Gln Ala 515 520 525 Ser Leu Ser Ile Ser Val Ser Asn Ser Gln Ile Gln Glu Asn Val Asp 530 535 540 Ile Ala Thr Val Tyr Gln Ile Phe Pro Asp Glu Val Leu Gly Ser Gly 545 550 555 560 Gln Phe Gly Val Val Tyr Gly Gly Lys His Arg Lys Thr Gly Arg Asp 565 570 575 Val Ala Val Lys Val Ile Asp Lys Leu Arg Phe Pro Thr Lys Gln Glu 580 585 590 Ser Gln Leu Arg Asn Glu Val Ala Ile Leu Gln Ser Leu Arg His Pro 595 600 605 Gly Ile Val Asn Leu Glu Cys Met Phe Glu Thr Pro Glu Lys Val Phe 610 615 620 Val Val Met Glu Lys Leu His Gly Asp Met Leu Glu Met Ile Leu Ser 625 630 635 640 Ser Glu Lys Gly Arg Leu Pro Glu Arg Leu Thr Lys Phe Leu Ile Thr 645 650 655 Gln Ile Leu Val Ala Leu Arg His Leu His Phe Lys Asn Ile Val His 660 665 670 Cys Asp Leu Lys Pro Glu Asn Val Leu Leu Ala Ser Ala Asp Pro Phe 675 680 685 Pro Gln Val Lys Leu Cys Asp Phe Gly Phe Ala Arg Ile Ile Gly Glu 690 695 700 Lys Ser Phe Arg Arg Ser Val Val Gly Thr Pro Ala Tyr Leu Ala Pro 705 710 715 720 Glu Val Leu Leu Asn Gln Gly Tyr Asn Arg Ser Leu Asp Met Trp Ser 725 730 735 Val Gly Val Ile Met Tyr Val Ser Leu Ser Gly Thr Phe Pro Phe Asn 740 745 750 Glu Asp Glu Asp Ile Asn Asp Gln Ile Gln Asn Ala Ala Phe Met Tyr 755 760 765 Pro Ala Ser Pro Trp Ser His Ile Ser Ala Gly Ala Ile Asp Leu Ile 770 775 780 Asn Asn Leu Leu Gln Val Lys Met Arg Lys Arg Tyr Ser Val Asp Lys 785 790 795 800 Ser Leu Ser His Pro Trp Leu Gln Glu Tyr Gln Thr Trp Leu Asp Leu 805 810 815 Arg Glu Leu Glu Gly Lys Met Gly Glu Arg Tyr Ile Thr His Glu Ser 820 825 830 Asp Asp Ala Arg Trp Glu Gln Phe Ala Ala Glu His Pro Leu Pro Gly 835 840 845 Ser Gly Leu Pro Thr Asp Arg Asp Leu Gly Gly Ala Cys Pro Pro Gln 850 855 860 Asp His Asp Met Gln Gly Leu Ala Glu Arg Ile Ser Val Leu 865 870 875 3 43950 DNA Homo sapiens 3 cgcggcgggg agggcagggg tgacgctcgg agaacagaga ggccgaaccc agagagcggg 60 ccgggacctg ataccgattt cccacccgtc ccctgccatg ggcgccggac gcctgccgga 120 gagggctccc ctccttaaag ggccagtggc ctccaagccc gacgcctgcg accggcggtg 180 ggtgatagtg tttcccctcc ctgtccagcc gagggaaaag ttaactttcc aggcttggct 240 gtgttcaggg aaggaactgg tctcgcctgc ctgccctcca tccctcacac catcccttgt 300 cccggaccct ggaggcggag gtccagcccc caactcggag gccccgggcc caccctcccc 360 ttccgccccc ggcccctcgg caggctccgc ccctctctga cgtcgccgag gcccgcgccg 420 attggtcgac tgcactgtcg ctccggacac ttcctcctgg gccgccgccg ccgccgccga 480 cttaaacttt ggagggggaa aaagagctac tggcgcctgg cgaccctccc tgccccccac 540 ccaaccccgc tccggcaacg cccccttcct cacggctccc gaccgaactt ttctccaact 600 tctgcgactc gtgagattcc cttctaccca ctccggccct cgggacccct ctgcccatcc 660 cctggccggt cgggtccctg cgaacccctt tatctctgga atccactcgg tccccgactc 720 agagactcct gccctccacc cccaaggtga attcccccgg gccgccttct gagtgggatc 780 ctcttcttgg agcactggat cctgggattc cctctgcccc cttctcaatc cctcctctag 840 ggaaggggcc tttgaatcgc gggctctcct gatccctgtg accccgacct actagatttc 900 ctctcaggct tcttggaatc tcaatcgctg ggacctccaa cccactactt ttctcctttc 960 tgatcttctg ggagccctgg attccgggcc tctgacccac tatagtgcct ttctctcctt 1020 cccaggaccc cgccatcctc aggtcccctc cgcctgccag atcttttctc ggatccccgc 1080 tctcccacca cctgctcacg agatcccgcg gatctagaac ccagggtccc ccggggcccc 1140 ccggccgggt cccgggtggg ctccaggcgg ccggtccccg gcctcccccc atggccaccg 1200 ccccctctta tcccgccggg ctccctggct ctcccgggcc ggggtctcct ccgccccccg 1260 gcggcctaga gctgcagtcg ccgccaccgc tactgcccca gatcccggcc ccgggttccg 1320 gggtctcctt tcacatccag atcgggctga cccgcgagtt cgtgctgttg cccgccgcct 1380 ccgagctggc tcatgtgaag cagctggcct gttccatcgt ggaccagaag gtgagggcgc 1440 aggctccctg gatccagctc ggggagaggt tgaaggaggg ggcgctggca gaggggtctg 1500 gggcctggtg tgcggaagag ggaggaagga gacctgagct ttgggtgatg gagggatagg 1560 gggcattgcc ccccttccat tgcccctctc cccaccatcc ctttgagaga ggactgggca 1620 ggggtggggt gccccagagg cctccccaaa tttcatgtcc ctgcatgtcg ttgttttctg 1680 cagcaaacag ggaggaaggg aggggccagc caggtgtaga gaggggagga aggggcagca 1740 gatgtcggcg gacctccacg tccaggccca tcccgggcct cccatttggt ggaaacagga 1800 gaaattgaac ccgggctggc catggtgatc cggtgacatg tgtgggtgca ggtgcttgag 1860 ttagctgcca ggggcaagtg aggtctcgga gcccaattct gccctcccct aagcctgaga 1920 tatgtgtgga ggggcaggca ctcctacaga ccctggggac tctattccct ttcctagtca 1980 cagtgctgtt agcctactct taattttgga caccagggtc cccagggtgg gcagctgggt 2040 gttatggcaa gaggaaacca ggtggaactc cacgtctaaa ccgtgaaatg ttaaaagaat 2100 agtgggcttc tgtgttggag tactggactg tagaaatgtt agaatattag aatcataact 2160 tgttggaata tgcatcctag gcaattaaat tgcccccatg ttcgtgttca aatattagaa 2220 ttctaggttt gtgaaatagt aaaacattaa aatgctggaa tattagattc ctagattgtt 2280 gaatcctaga aagttaaaat gttagaattt tagaatgctg gatggatgag gtccttgaat 2340 gctaaagaat tcaaagagca cagtcctagc ttgtcagact cctagaatat taaaatatta 2400 gattaccgct tatttaggtt attgaaatcc taaaatgtat agtgatacca ggtaggaatc 2460 tagaatgtat aattctataa tgtgagcatg ttggagtccc aaaatatcca aattccagaa 2520 tcttttcaga ctcctggaaa tgaatccttt gggcatcaga gaaacgtggg gaactgggcc 2580 agctccccca ttctacagac aaggaaactg aagcttagag aaaaacttcc caaggggtca 2640 gggccaaggc agtcctggtc ttctgtggac tctctcttag cagtgagaac tgatagggtt 2700 ttgcccacca aatgcctaaa tcccgcaggc ccagctcacc accccaactc agcccacttc 2760 atgggaagct ggtggcagtg ggggtacggg ggcagattgt cccttgggtg aacttctttg 2820 tccagtgctc aagtccccag cctgccccgc tcaggcttca ccccagtttt atttttctgc 2880 caggtccagg tgtgttaggg ccgcgtacct tccttcccga ggccccaccg gggcagtttc 2940 actttctgtt ctactaggtt tcatttcctg cccccaggcc cccaaagctg aggacccaga 3000 cacctgggtc ctttgagcat tgggtggcag gcgccctcct tatctccagc gccctcgagt 3060 ccaagtcccc cggccccccc cccccacttt cccaggagcc ccgaaaagtc ctccttccag 3120 ctcgccccac cccagtgctg ggcctggagc caggtaactg ggacaacaat agacagatcc 3180 aggaaggaag ctggggggcg ggtgtgtgag cctggggagg aggcacaggg gagggagtgt 3240 tcattcagca tcccctccca cctccgccag gttccggaaa attcgaggtg tccacgctcc 3300 cggagccact ctccctccca ccccagctcc cccttccagc caccaaaccc acgccggcgc 3360 cccctccccg tacaattggg gcgctggcat cctgcccggc tcgcgctggg gttgggaggg 3420 ggcaggcagg aagcgagggc ctgcggggtc tctgcgtttc cgggggaaac agccggccct 3480 gccctgggag ggtcacagtc cgcccgctgc tgaaggcggc tctgagcttt tccgtcgcca 3540 catccctctc ccgcccctca gttccctgag tgtggcttct acggccttta cgacaagatc 3600 ctgcttttca aacatgaccc cacgtcggcc aacctcctgc agctggtgcg ctcgtccgga 3660 gacatccagg agggcgacct ggtggaggtg gtgctgtcgg gtgagaggtg gtggccggcc 3720 tgggggcggg gcctcgggtg ggggcggggc atctggggga ggagagggta gggggagtta 3780 gaagtcagga gaggccgggt gtagtggctc acgcctgtga tcccagcact ttgggaggct 3840 gagctggagc tggggggatc gcttgagccc aggagttcga gatcagcctg ggcaacatag 3900 tgagattcca tctctacccc tttctctccc tctgaaaaaa aaaaataagg agagttgggg 3960 gcttctggaa gatggttaca gagtggggtc atgaaggcgc tctttaggga ctggtctaaa 4020 ctttcattta tggattagga tgctagtgac acgctttgta cagtttgaaa attcattgag 4080 ctgtgcactt gtgatgtgcg gcctttcctg aacatatgtt atacttattt atttataaaa 4140 ctagtcaagt gcagtagtta gaagggggaa aagaggagaa gaaggagttg gatctgtaac 4200 tgactgtgtt atgcttaaat ataaaggtaa aaaatgggcc agctgcagtg gctcacacct 4260 gtaatcccag cagtttggga ggctgaggtg ggaggatcgc tggagcccag gagtttgaga 4320 ccagcctggg caacataagg agaccccatc tcttaaaaaa aaaaaaaaaa aaaaaagtta 4380 accgggcgag gtggcacacg tctgtagtct cagctacttg ggaggctgag gtgggaggat 4440 ttcttgagct taggagtttg aggctgcagt gagccacgat catgtcactg cactccagcc 4500 tgggcaacag agagagaccc tatctctaaa aaagaaaaaa agtagaaaaa gaaaaaaaaa 4560 agttatgatg tccatggctc ctgccacgaa aatgctaaat taaatcagaa tctctgcaaa 4620 gtgagatgga atctgcacat cagtattttt aaaagccccc aggtgatttt ctaagacaca 4680 gccagaagcc agttcatcca ctcactattc cagtagtata gatgggcatg ctctcagcac 4740 cttagagcag tctatggccc ttggtccctc ttgagggtgg gggcagctgc ctttttcatg 4800 gctgtcttcc ctgctgctcc ggcatactgc agtgcccagt gaaaccggct caatgaatga 4860 atgacagaag tctggattta cacctttagt gaccttgttc aggctttaag tactctttca 4920 tatcataagc tggcctcact tgaattttta tcttcattgt tgtctctccc ctaaacctga 4980 gttttgtttt gtttttgtca tttttattat tttttgtttt tttagacgga gtctcgctct 5040 gtcacccagg ctggagtgca gtggcgcaaa ctcagcttgc tgcaacctct gcctcctggg 5100 ttcaagcgat tctcctgcct cagcctcccg agtagctggg attacaggcg cctgctacca 5160 cacgtggcta atttttgtat ttttagtaga gacgggattt caccttgttg gccaggctgg 5220 tctcgaactg ctgatcttaa gtgatctgcc cacctcagcc tcccaaagtg ctgcgattac 5280 aggtgtgagc caccgctccc ggccctgtta ttttgttttg aggcagggtc ttgttctgtc 5340 acccaggctg gaatgcagtg gcatgaccac cactcactgc agcctctacc tcccagactg 5400 aagcaatcat cccgcctcag cctcctgagg tggctggact ataggcatta caggcatgca 5460 ccaccacact gggctttttt tttttttctt ttttttagac agaatcttac tctgtcaccc 5520 aggctggagt gccgtggcat gatcttggct cacggcaacc tctgcctccc gggttcaagc 5580 aattctcctg cctcagcctc ctgagtagct gggattacag gcacgcggca ccaggcctgg 5640 ctaatttttg tatttttagt agagacgggg tttcatcatg ttggccaggc tggtttcgaa 5700 cttctgacct caagtgatcc gcccacctgg gcctcccaaa gtgctgggat tacagatgtg 5760 agccaccggg caccgcctat ccatgttctt ttttgttgtt ggtggtggta tttttaatta 5820 aaaatttttt aatttggtaa aatatacata acataaaaat tactatttta ggccgggtgc 5880 agtggctcac gcctgtaatc ccaacacttt gagagaccga ggcgggcaga tcacctgagt 5940 cgggagtttg agaccatccc tggccaacat ggtgaaactc cgtctctact aaaaatacaa 6000 aaattagtcg ggtgtggtgg cgcatgcctg taatcccagc tactctggag gctgaggcag 6060 gagaactgct tgaacccggg aggcggactt gtggtgagcc gagatctcac tactgtactc 6120 cagcctgggt gacagagtga aactctctaa caaacacaaa caaaaaagcc cacaacattt 6180 taagcacttt taagcgtaca gttcagtaat ttaaagttca cgcacactgt tgtgcagccg 6240 gtctccagaa ctgttgtcat cttgcgaaac tgaagctcct tgcccgttaa acaactcccc 6300 aattcccgct ctgtccctgc ccagggcgta gggatatatg tgttttgttc aggggtggag 6360 ctgggatttg aacccaggca gaatgtagta tgagagcaaa tgaaggaagg aaggaaagat 6420 cacaccttgc ggctgggagc actgtgagaa atcagggaac gtggggtctg gaaaagcttt 6480 ggcctacccc gcctcaagca tccaccccta ttttccgcct acagcctcgg ccaccttcga 6540 ggacttccag atccgcccgc acgccctcac ggtgcactcc tatcgggcgc ctgccttctg 6600 tgatcactgc ggggagatgc tcttcggcct agtgcgccag ggcctcaagt gcgatggtga 6660 gagctaaagg gttgggggcg gggcctgggg cggggctctg caccgggggc ggagcgtaat 6720 ggtcctggca cggggacagc gtggggagga ggagcgggtc tcagagctgg gggcgcagcc 6780 taggaagtaa taatgggaag aaggatgggc ccagaagcag agcttgggga aggagtggtg 6840 gggctgggcc ggggctcagg tctaggggcg gagcctagga ggtggagctg ggagggacaa 6900 gtaggggctt aagaacagag cctaggggag cagaagggtg gcgggggaag agggtggggc 6960 ctctatcagt tagggatcaa gcagagaaac atccaggagg agatatatat tgagatattt 7020 atatgcaagg aatcagctta cagaattgtg tgggctggct aggcaactca aatctggctg 7080 ggcacagtgg gggaggccag taatcccagc actttgggag gcaaaggtag gtggatcact 7140 tgaggccagg agttcaagac cagcctgggc aacatagcaa gactctgcct gtacaaaaaa 7200 taattagcca agcatggtga cagacacttg tggtcccagc cacttgggag gctgaggcgg 7260 gaggatcact tgagcctggg agctcgacac tgtagtgagc cctgattgca ccactgcaca 7320 ccagcctggg tgacagagcg agaccctggc tcaaaaacag gaaaaaggcc ggacacggtg 7380 gctcatgcct gtaatcccag cactttggga ggccgaggcg ggtggatcac gaggtcagga 7440 gattgagacc ctccctggct aacatggtga aaccccgtct ctactaaaaa tacaaaaaat 7500 tagccggacg tggtggcaca cgcctgtagt cccagctact tgggaggctg aggcaggaga 7560 attgcttgga cctgagagga ggaggttgca gtgagccgag attgtgccac tgcactccag 7620 cctggtgata gagtgagact ccttctgaaa acagaaacaa aaacaaaaca ataaaaagaa 7680 aaagaaaaaa aaatccatcc tatcaggaag ggcaagtggg aactcaggca caagctgaag 7740 ctgatgtcca caggtggaat ttcttcatcc gaaaagtctc tgatctgctt tttaaaacat 7800 tcagctgatt gaatgagacc cacctagaac aagcaggatc acctctccca cttacagtca 7860 gctgattatg gattttcatc acatccagaa aatacctcca ctgggccggg tgcggtggct 7920 cacgcctgta atcccagcac tctgggaggc cgaggcaggt gaatcacctg aggtcaggag 7980 ttcgagacca gcctgtccaa catggtgaaa ccccgtctct actaaaaata caaaaaagcc 8040 ggcgtgttgg tggacgcctg taattccagc tactcgggag gctcagtcag gagaatctct 8100 tgaacccggg aggcagagct tgcagtgagc tgagattgca ccattacact ccagcctggg 8160 caacaagagc aaaactctgt ctcaaaaaaa tgaaaagaaa agaaaatacc tccatggggc 8220 cttctctccc cagttcttcc tggagtcggg gaaaagctgg gttgagaagg tgaaaagaaa 8280 aaacaaacct tgactgggca cagtggttca cacctgtaac cccagcactt tggaggctga 8340 ggcaggcgga tcatgaggtc aagagattga gaccaccctg gccaacatgg tgaaacccca 8400 tctctcctaa aaatacaaaa attagcgggc gtggtggcat gtgcctatag tcccagctac 8460 ttgggaggct gaggtaggag aatcacttga acccaggaga cagaggttgc agtgagccga 8520 gatcgtgcca ctgcactcca gcctggcaac agagcgagac tccgtctcaa aaaaaaaaaa 8580 acaaaaaaaa aaaacacaaa caaaccaacc ttcatggcaa catctagatt agtgtctgaa 8640 taactgtgga tctcgcctag ccaagctgac acattaacat gactatcagg gtccatctct 8700 tgtcaacctg gcacctgtct tagtttgtca gggctgcctt aacaaaatac caccctgcgt 8760 ggcttaaatg acagacattt acttctcaaa atccctggaa ttgtgagagg ctggaaagac 8820 aaagatccag attctggcag ggttctgttt ctggtgtagc ctgctttcct gccttgcaga 8880 gggccatcat ttcactgtgc gctcacatgg gacacggaga gagagatccc tggtatctct 8940 tccctttata aggaaggcca ggcatggtgg ctcatgccta taatcccagc actttgggag 9000 gatggtggat cgcttgagtc caggagttcg agaccagcat gggcgacatg gtgaaacccc 9060 gtctctaaaa aatacaacaa attggccagg catggtggtg catacctcta gtcctagcta 9120 ctcaagaggc tgaggtggga ggatcacctg ggcctgggag gttgaggctg cggtgagccg 9180 tgatcatgcc actgcactcc agcctaggtg acagaacacg attgtctcag gaaaaaaaaa 9240 aaaaaaaaaa aaaaaagggt caccagtccc attggattac agccacactc tttcggcctc 9300 aattaacctt aattacctcc ataaaggcac cgtctccaga tatagttgca ttggaggtta 9360 gggtttcaac ataagaattt tgggggagac acagacattt agtccataac agcacccata 9420 catatctcct taaatcatag tttaaaaata tacaggtttt cttttttgga gacagcgtct 9480 cagtctgtca cccaggctgg agtgcagtgg cgcgatctca gctcaccaca acctccactt 9540 cccaggctca agcgattctc ctgcctcagc ctaccgagta gctgggatta caggcacaca 9600 ccattactgc ccggctaatt tttgtatttc tagtagagac ggggtttcac cacgttggcc 9660 aggctggtct tgaactcctg acctcaaatg atccacccgc cttgccctcc cacagtgctg 9720 ggattacagg catgagccac cgcgcctgtc caaaacatac agttctttaa gccaagatgt 9780 ctcaaggttc agcccaagtg tcaagatcta tataggtcct ctgtccctgt tattcatgct 9840 tctgagtgag aatgttgaaa tcggggctct gcctacagat gaaggccatg tacctgcatt 9900 ggctatgagg acagatgaca ggtgaggacc atccattctg tgatgagacc ctgtggctcc 9960 atttttttgt gtgtgtgaga cagagtcttg ctccgtcacc caggatggag tgcagtggcg 10020 tggtcttggc tcactgcaac ctctacctcc tgggttcaag caattctcct gcttcagcct 10080 cccaaatagc tgggattaca ggtgcgcacc accactcctg gctaattttt gtatttttag 10140 tagacggggt ttcaccatgt tggccaggct ggtttcaagt aatccaccct cctcagcctc 10200 cccaagtgct gggattacag acatgagcca ctgcgctggg ccccatgcgc ctccattttt 10260 gtatggtgtg ccctgcaatt agagccatat tcttggatgt tccattgggt attaggtctg 10320 agacagcatc tctagctccg tgggtgccac gcttgtacag aaatcctgat tctgggccag 10380 gcacggtggc tcacacctgt aatcccagca ctttgggagg ccaaggcggg cggatcatga 10440 ggtcaggagt tagagaccag cctggccaac atggtgaaac cctgtctcta ctaaaactag 10500 aaaaattagc tgggtgtggt ggcgggtacc tataatccca gctactcggg aggctgaggc 10560 aggagaatca tttgaacctg agggggtgga ggttgcagtg agccgagatc ataccattgc 10620 actccagcct gggtgacagg gtgagactcc gtctcaaaaa aaaaaaaaaa aaagaaatcc 10680 agtttctcca atatcctgtg ttccagatca tcatgcagtc caaagtatac ttgtattatt 10740 taaggactct aggcctgcag atactgattc agtgcattaa aagctcttat aaatattgcc 10800 atcgtccaca caccatatcc aactcttgag gtctcagcat atgcagtctt tgtcatgata 10860 cagccctggt gtcatcaagt cctaatgggt tatcagcaca gacttcactg gtgcagcatc 10920 acagatgatg gtcccagttc ctatggtggc aagagaaccc caaatgacta cattccgaca 10980 ggagtttaac tctatcctga gactcattct gagagttata gataagattc tgaaattctg 11040 gaaggcacat gagtgattca aggccaacac tgggaaatgg ttcctgtgtg caaagaccat 11100 ttgccctgct gaagctcttc ttgcagggcc aacaccgttc tccaagcttg cctccgtgat 11160 tacagcatgc agccaagaca gtgcctacaa tgaggaggtg tggaactgga aagcctggag 11220 caggcgggta ccagaagggc tcccaaaggc tggaggaaca ttcttcactc cagaatagaa 11280 agcgatcctg gaatcgtttg gaatcactgg agatgtatta gagcacacat acagaacgtc 11340 cagtgggaaa cagggagttg agctgatttc tccatggatg aggattttaa aagataaaat 11400 aggcagggca cagtggctca tgcctgtaat cccaacactt tgggaggctg aggtgggagg 11460 atcacttgag cccaggagtt caagaccagc ctgggcaatg tagcgagacc ccatctctac 11520 aaaaaaataa aaataaaaaa attatctggg catggtagtg tatgtctgtg gttctggcta 11580 ctcaggaggc tgaggcagga ggattacttg agcccaggag ttgaaggctg cagtgagcta 11640 tgattgtgcc attgtgcttc agccgggggt acagggagat cctgtctcta caaaataaaa 11700 taagacaata agaagtcata cttctgccta gtatggtaca atggacctga gtacaactga 11760 gaactctttt tttttttttg aaactgagtc tcgctgtatt gcccaggctg gagtgcagtg 11820 gcgtgatctc agctcactac aacctctgcc tcctgggttc aagtgattct cctgcctcag 11880 cctccggagt agctgggatt acaggcgtgt gccactacac ccggctaatt ttgtattttt 11940 agtagagatg gggttttgcc atgttggcca gtgtggtctc aaactcctga cctcaagtga 12000 tccgccggcc ttggcctccc aaagtgctgg gattacaggc gtgagccacc atgcgtggcc 12060 cacactacta agatttaatc acactactta gggattgcct ggattccagg tctacagaaa 12120 agagaaagtg gggtacaggg ggtgagcaga cctggaggga tagtgacctt aggggtgggg 12180 gtgaggagag gcattttctt ttggaaagtt ggggttgggg aaagaggggg aaccaaaggg 12240 gcctcagaaa aaggaaggtc agggttagaa gggggaacag gtgtctctag ggagatggac 12300 aggagttttg gggaggacta gaaggaggtg cttaccatag aggactgggg ctgggtcaga 12360 gctttggcgg ggacttttga ggcatccatt gttgcagtgg gaaaaggtgg ggtgtgaggc 12420 gcgttcaggg cctggggggc agatggggtg atgtcggggc tacaagctgg aactaggggt 12480 ggagctttgg agggaacctt tgaggtatcc cttgttggag tgggaaaatt ttgggtgtga 12540 ggcgtgttca gggtctgggg gacagatggg gtgatggcag ggctacaagc tgaaactggg 12600 gacagagctt tggggggagc ctttgaggtg acccttgttg gagtgagaaa aggggtgtgg 12660 gtgtgttcag ggtctggggg acagatgggg tgatggtggg gctacaagct ggaacttggg 12720 gcagaactct aaggaggggt gggcctgaag gggctgatac acttacggat agtagtgcct 12780 tttggaggag atcgtgctgg cggggggtga tgggacagga ccaggtgaga gattgggtgg 12840 aaagggcaca acttctcaag aagagaccta ggaggggcag acgccatgtc tcttactctc 12900 tggcgccccc tgcaggctgc gggctgaact accacaagcg ctgtgccttc agcatcccca 12960 acaactgtag tggggcccgc aaacggcgcc tgtcatccac gtctctggcc agtggccact 13020 cggtgcgcct cggcacctcc gagtccctgc cctgcacggc tgaagagctg gtgaggagat 13080 gggggatggg acgggttggt ggctaggggg gtgacttggc ccaggcatgg ggccaacgca 13140 ctgatgtgtc ccctccattc ttgccaatga cagagccgta gcaccaccga actcctgcct 13200 cgccgtcccc cgtcatcctc ttcctcctct tctgcctcat cgtatacggg ccgccccatt 13260 gagctggaca agatgctgct ctccaaggtc aaggtgccgc acaccttcct catccacagc 13320 tatacacggc ccaccgtttg ccaggcttgc aagaaactcc tcaagggcct cttccggcag 13380 ggcctgcaat gcaaaggtta gctgggcctg tcggggagga cagtacaggg tcagaacctc 13440 cttcccgccc caacctggtc ttgtggcagg acacaaggat ctgagccttg ggaccccagg 13500 gcctcagaag gggagggccc tgaatcctag tgttctggga cctttggaat tctggaatct 13560 tagaacctca gttgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgttgt gttgtttttt 13620 gaagacaggg tgtcactcta tcacccaggc tggagtgcag tggcgcaatc acggctcact 13680 gcagcttcaa cctcttgggt tcaagtgatc ctcctgcctc agcctcccaa gtagctagga 13740 ctacaggtgg tgccaccaca cccagctaat tttcttttct tttttttttt tttgagacgg 13800 agtctcactc tgtcgcccag gctggagtgc agtggtgtga tctcgggctc actgcaaact 13860 ctgcctcctg ggatcaggac attctcctgc ctcagcctcc tgagtagctg ggactacagg 13920 cgcccgccac catgcctggc taattttttt gtatttttag tagagacggg gtttcaccat 13980 gttagctagg atggtctcga tctcctgacc ttgtgatcca cctgcctcga cctcccaaaa 14040 tgccgggatt acaggcgtga gccaccgcgc ctggccacac ccagctaatt tttaaatcat 14100 ttgtagagag aaggtatcac tatattgttc aggctggtct tgaactcctg ggctcaagca 14160 atcctcctac ctcggcctcc caaagtgctg ggattacagg tgtgagccac cgcgcccagc 14220 tgaacctcag tctttagaac cttggaatcc tagattcata acgtgcttag catggaattc 14280 taaaactgta gaacctgaga attctagaat cagaaccata gcattcaaga attccgaatg 14340 atagaattca gctaaaataa caacagaact ttagattaca catcttagat ctcccaagtt 14400 atagactctc agagcatgag aattttggaa ccatgggatt tgagggtaat agaaacatag 14460 gcacatcaaa tttgagagtc ttagacgtct agaatcatat aagcttgaaa ccatcgtaac 14520 ctagaatcct ggaaattcta gactcccaga actttgaaca atcaaattct agaatccagc 14580 caggtgtggt ggctcatgca tgtaatctca gcactttggg aggccaaggt aggtggatca 14640 cttgagccta ggagtttaag accagcctgg gcaacatggt gaaaccctgt ctctacaaaa 14700 aaaattaaaa attagccagg catggcagca tgcatctgtg gttccagcta cttgggactc 14760 tgaggaggga ggattgcttg agcccaggag gttgaggctg cagtgagcca tgattgtgcc 14820 actgcattcc agcctgggtg acagagcaag aacttgtctc aaaaaaagaa aaaaaaaaat 14880 tctagaacct cagaagccta gatccacata aacttagaaa catccaattc aagaatttac 14940 tggaacaatc aaattctaga atcttagaag cctagagcta aagaagcata gaaacatcaa 15000 attctagaat cttgtatgta tagaatccta gaaccttgga atctgcagat tctggaggta 15060 gagaagccta gaattgtaga accctagaac tgtcaaattt tagagtttag atatataaca 15120 ccctaaaatc ttggacatta aagagtctta gaagtgttga ctcatagatg tctagagttc 15180 tagaaacttg gacatcaaac tctgaagcct tagaaatacg gaatcaggtc aggggcagta 15240 gctcacacct gtaatcccag cactttggga ggcttaggtg ggtggattgc ttgagcccag 15300 gagttcaaga ccagcttgta caacatggaa agaccccatc tctacaaaaa atacgaaaaa 15360 ttagccaggc atggtagtgc gtgcctgtag tttcagctac tcaggaggct gaggtgggaa 15420 gatcgcttga gcctgggagg cagaggttgc agtgagccga gatggtgcca ttgcacactc 15480 tagtctgggt gacagccaga ctgtttctta aaaaaaaaaa aaaaaaaaaa aaaccagaat 15540 catagaacct tcataaaata ggtttttagt aaactctaga atcttcgatg tatagtgtcc 15600 ctagaaccgt ggaaacactg aactctacag caatggttct cgaccagggg ccgttttgct 15660 cctaggggat gtttggcaag ggttggagat ggttttgttt ggtacgctgg gatagtgcta 15720 ctggcatcca gtaggtagaa gtcagagatg cagctaaaca tcctacaata cacagagcaa 15780 gtgccctaaa acaaggaatt atcctgggca ctgtgttagt gtcacgggtt gaggaaccca 15840 gccctagggt gttcagagtc tggagtcaca gcacattaga accaataaca cacacacaca 15900 cacacacaca caagtcgggc gcggtggctc acgcctgtaa tcccagcact tttaggaggc 15960 caaggcaggt ggatcatctg aggtcaggag cgcgaaacca gcctgaccaa catggcgaaa 16020 ccccgtctct actaaaaaca caaaaaaatc agctgggcgt ggtagtgggc gcctgtagtc 16080 ccacgcccag ctaatttttg tatttttagt agagacgagg ttttaccatg tagggcaggc 16140 tggtttcgaa ctcctgacct caaatgatct gctctccccg gcctcccaaa ataccgagat 16200 tacaggcggg agccactgca cccagcagtc gtcgggattt tgagtctagc cctcctactt 16260 aatcaagacc cccccgatgg ttgggaaaac tgtggctgaa agtgggaaaa tgaccagggc 16320 agcagcagcc agtgttctta cccagacagc aagagtagac tcttttgagc ctgaggctta 16380 gggtcaaggt tcaagccttc caggtaacct ctcttcccct tctcacccgt tcccttgttc 16440 cctgtcctac cagactgcaa gtttaactgt cacaaacgct gcgccacccg cgtccctaat 16500 gactgcctgg gggaggccct tatcaatgga ggtgagaggc tggggggatg ctggggagaa 16560 aggggaaggg gcaggactgg gtggagaccc ctctgatgcc tccgtcccca cagatgtgcc 16620 gatggaggag gccaccgatt tcagcgaggc tgacaagagc gccctcatgg atgagtcaga 16680 ggactccggt gtcatccctg gctcccactc agagaatgcg ctccacgcca gtgaggagga 16740 ggaaggcgag ggaggcaagg cccagaggta tacacagaac cctccaagag accctggggg 16800 aagaccctcc tgcacagtga acctcaattt ctttttctct acaatgggct gacatcacct 16860 catatttata aattttccca gttcctgagg caaacctttt aaagcactac aatttttttt 16920 aaataatttt ttgtttgaga cagggtctcg gtctgtcgcc caggctggtg cagtggtgca 16980 gtcttgactc actgcagcct cgaccacctg ggctcaagcg atcctgccac cttagcctct 17040 cgagtagctg ggaccacagg ctcgtccacc acacccagct aatttttgta tttctgtaga 17100 gacagggtct accctatgtt gcccaggctg gtcttgaact cctgactcct gagctcaagt 17160 gatccacccg cctcagcctc ccaaagggtc ttgctttgtt gcccactgga gtgcagtggt 17220 gtgattgtgg ctcactgtaa cctcaaactc ctgggctcag gtgatcctcc tgcctcagcc 17280 tcccgagtat ctgggactac agggatgcac tgctatccct ggctaatttt agacggcgtt 17340 tcgctcttgt tgcccaggct ggagtgcagt gatgcaattt cagttcattg caacctctgt 17400 ctcctgggtt caagcgattc tcctgcctca gcctcccaag tagctgggac tacaggcacc 17460 cgcccaggcc cagctacttt ttttgtattt ttagtagaga cagggttttg ccatgttggt 17520 caggctggtc ttgaactccc aacctcaggt aatccacctg cctcggcctc ccaaagtgct 17580 gggattacag gcatgagcca ccgcgcctga cctatattcc tcttcttttt tttttttttt 17640 tttttttaag atagggggtc ttgctatgtt gcccagggtg gtcttgaact tctgcgctca 17700 agcaatcctc ccacctcagc ctcccaaagt tctgggatta caggtgtgtg ccactgtgcc 17760 cccagcctac acatttttaa actatacacg gagttcatac ttagtcagct ccactggaat 17820 gtgagctcag gtgcatgagg gcaaggatat tttctgccct cccaggtgcc taggacagga 17880 ctggctcaga tcaggcactt cctatctggg tgtggcgtga atgtttattg agaaagcaca 17940 gttcacacag gcgctggagg gtgacagccc agatcccagc tctaccactt cacttgctag 18000 gcgcttccct gtgtgccacg gtttcctcct ggggcgatga ggtacctacc ccacggggtg 18060 ataaacctgg ggtaggggta agggggcacc ctcacaggtg cactggaaaa tatttaatga 18120 gcacctgctg tgttcaagca cacagctatg aacaaaagag gtaaaagtct gcccttctgg 18180 agctgactgc ctcagtgggg agacagctaa taaatgcatc catagcatcg ggtattggta 18240 atggtgataa aaacaagagg agatggagaa tgggggacat gctatcttag ggtccttcaa 18300 ggagacctcg ctgaggaagt ggcagttgaa gggaggggag ggaaggagcc ttgtggggct 18360 ctgggggaaa aggcttccag gcagaggcaa cagcgagtgc aaaggccctg gggtggaggc 18420 accgtgttcc agggacagca aagagaccca tgtagctgca gcagggaggg cgaggggaag 18480 agggttggac agaaagggga tgggtaagcc agtcacagtg acgacagagt gtttcctgcg 18540 gtgcctccca acccaagcag cctgaagccg caggttccct ttctcccacg tctttcctgg 18600 gaatgcctag taacaccgtc atacactgtc aagagttgga ccttgaggga ttgggggtgg 18660 cgggtgtggg gagaggcagc ccatttcaca gatggggaaa ctgagtctca ggcaaagaga 18720 tgtgatcaag gccacccagg ttctgatcta gcacagggat ccagagattg ttggttccag 18780 agttgagcaa gtcacttaat ctctcaaatc tcaaactcct gacctcaagt gatcccccca 18840 cttctgcctc ccaaagtgtt gggattacag gcatgagcca ccatgcccag caggccactt 18900 aatctctgta gaccttcctt actgtactaa cagcatctgc acaaatgagg gaggtgaggc 18960 ccagagaggt tgaatcactt acccagtgtc acacagctgg ctccacaatt gctggactaa 19020 ataccaatta gcacttactg gaggtcctct gtatgccagg cactgtacta agctccgtag 19080 aaaggtttcc attcctcata gcatcccctt tgggtggaca aactgaggca tgaagaggtt 19140 aggtaatttg ctaggcagcc tgacttcaga aaggcctact acagaagccc tctcaagaat 19200 ctccttctgg gccagcgtgg tggctcacac ctgtaagcac tctgggaggc cgaggcggat 19260 ggatctcgtg aacggattct aagggtggga ctaggggcag gagttaggga aggagttgag 19320 gcaaagagtt cgagaccagc ctggccaaca tggtgaaacc tcatcactac taaaaataca 19380 aaaattagcc aggggtggtg gcgtgcacct aatggtcacc gtgattgtcc cggccactca 19440 ggaggctgag gcacgagaat cgcttgaacc cgggaggcag aggttgcagt gagccgagat 19500 cgcaccactg cattccagcc tgggtgacag agcgagcctc ttaaaaacaa acaaaaagca 19560 actcccgggt gtgtgttggg gggaaaatgt caaaacaaac caaacaaaca aaaacagtcc 19620 ccaactccct agtttcccag agatgccccc tgcattccca agcagcatgg tcactttctg 19680 catgtgactt ctcacccctt cctcttcctt cgcagctccc tggggtacat ccccctaatg 19740 agggtggtgc aatcggtgcg acacacgacg cggaaatcca gcaccacgct gcgggagggt 19800 tgggtggttc attacagcaa caaggacacg ctggtgagtg gccggggcgg ggccgggtac 19860 ggcggagcga aggctggaag aggggcggct cagcttgagt aggcggggct aggtgggtgg 19920 ggctggagct aggcgcgagc ggggccagta gtgggctggg ccgtgctgga ggcggggcta 19980 gaattagaag tgtgggctgt aagggtggga ctacgggcag gagttaggga agacccgggg 20040 ctcagggcaa ggtcaggggc ggggctagag ttaggggagg agcttggctg gaggaagagg 20100 gctaagtggg ggcgagtctg gggttagggc gtgggggctg ggctagggtt aaggctaggg 20160 gcggggctgg ggttagggcg tgtggtgggg tggggttacg gcgtggggta ggtgctagag 20220 ttacggcgtg cacgtggtgc tccaggcacc tggagcccca agcagctcca cgggataggg 20280 actgggcagg aaagtctggc ggttcacgtg actcttcaaa catctctgca gagaaagcgg 20340 cactattggc gcctggactg caagtgtatc acgctcttcc agaacaacac gaccaacaga 20400 tactataagg taagcctccg ggctttcagc tccctcggac ttcccgctgt gcccacaaac 20460 tttcccacac ctcctcctac ccccagttac tccagacaga tcctgcaaat cacaccctct 20520 gcccaccccc agcctccctg cttccagctc atcagcaagt gctgcccatc cgattctggc 20580 cccaccactt tccagccagg gggactccgg gcaggttccc ttacttctca gtgcctcacg 20640 cttctcacct gcaaaatgcc tcaaatgcta atactcacct cagggctggt gcgagaattc 20700 aaagagccaa tccactaaac caattggctt aaggcgtggt atatattaag ctcccagtaa 20760 ttctaaggct gttctcacta ttcctttatt ttttgttatt tatttatttt ttgagacaga 20820 gtctcactct gtcgcccagc tggagtgcag tggcgcgatc tcggctcact gcaacctccg 20880 cttcccgggt tcaagcgatt ctcctgcctc agcctcccac cctaggacta caggtgaatg 20940 ccaccacacc cagctaattt ttgtattttt agtagagacg gggtttcacc atgttggaca 21000 ggatggtctt gatctcttga cctcatgatc tgcccccctc ggcctcccaa agtgctggga 21060 ttacaggcat gagccaccgc acccggcctc actatttctt tataattaat gtattgcatt 21120 gtgtgcgtat tcgtcaccac ctcccatgcc cacactgtgt cccagccact gtcttccacc 21180 tggatggttt cagccttctc cttgcagggt ccttgcttct gacctcacaa cctctgtcat 21240 ttcccccaca gccaggggga gtcttcatta aaaccgtcaa accccccagt ggctcccatt 21300 gtcttagaag taataaaacc tggtactcca gctgttacct gccctggaag cgtcttcctt 21360 gaactttcca tggctggttc cttatcatct tcccattttg ctcagaccac accatctaaa 21420 atgctgtcct tggccaggcg tggtggctca cgcctgtaat cccagcgctt tcagaggccg 21480 aggtgggcgg atcacttgag atcatgagtt cgaaaccagc ctggccaata tggtgaaacc 21540 ttgtctgtac taaaaataca aaaattagct gggcatggtg gcgggtgcct ataaccccag 21600 ctacttggga ggctgaggca ggagaattgc ttgaacctgg gaggtggagg ttgcagtgag 21660 ctgagatcgc gtcactgcac tcctgcctgg gcaacagagc aagactccat ctcaaaaaaa 21720 taaaataaaa taaaatataa tgctgtcctc accatgcccc cccgacgtgt ccatgtcatc 21780 acctggtttt atgggctgcc taagtcattc attctttcct ctctcctacc tccctccttc 21840 ctcttttgac acgtttccca ccccatagtc cctgtgcctt ctgtcccgcc tgggtcccct 21900 cagcctcctt cctggttctc tgtctccatc tcattctatt ccatctgccc tccgcacaca 21960 agcggatgat gctcaaaagc cttcagtggc ttcctagggc ccttggacaa agcccaggct 22020 cttccttgtg gcccgcaaag ccctgtgtgg cctcatttcc tccatttatt atcaaacgtt 22080 tatttttgag acggagtctc gctctgtcac ccaggctgga gtgcagtggc gcgatcttgg 22140 ctcactgcaa cctccgcctc cggggttcaa gtgattcttc tgcctcagcc tcccaagtag 22200 ctaggattat aggtgtgcca ccacgcctgg ctaatttttg tatttttagt agagatgggc 22260 tttcaccatg ttggtcaggc gggtctcgaa ctcctgactt tgtgatccgc ctgccttggc 22320 ctcccaaagt gttgggatta caggcatgag ccaccatgcc cagcccattt atttattttg 22380 agacaggctc ttgccctgtc tcccaggtgc agtggcatga tcatggctca ctgtaacctc 22440 tgcctccctg gctcaaatga ttctcccacc tccacagtag ctgggattac aggtgcgcac 22500 caccacacct ggctagtttt tttatttttt gtagagatgg gggtctcatt gtgttgctct 22560 ggctggtctc aaactcctgg gctccagcga tctgcctgcc ttggcctccc aaagtgctgg 22620 gattacaggc ttgtggcacc atgcctaatt tttaaatttt ttgtagagct ggggtctcac 22680 tgtgttgccc aggctggtct tgaactcctg ggccatctgc ccacctcggc ctcccaaagt 22740 gctgggagta caggcacgag ccaccacatc cggccatcaa aatgtttatc aagcttttac 22800 tatgtccagg caccgcccca tgtgatgggg atacagcttg gcttttgagc atagcctttc 22860 cttagggcct ttgcacatgc tgttccccta ctcccttgcc aactggctgc ttcttacctt 22920 tctggtctct gcttcaatat cacttctgcc agtaattagt attattatta ttatttttga 22980 gacggaatct cactctgtcg cccaggctgg agtgcagtgg tgcgatcttg gctcattaca 23040 accaccgcct cccaggtgca agcgattttc ctgcctcagc ctcccgatta gctgggatta 23100 caggcgcaca ccaccacgcc tggctaattt ttgtattttc agtagagacg ggattttgcc 23160 atgttggcca ggctggtctc gaactcctga cctcaagtga gctgcccacc tcggccttcc 23220 aaagtgttgg gattacaggc atgagccacc gcacctggcc tctgccagta attataaaag 23280 aacagtgaga acaggcttag aattactggg aacttgtctg accactgtgc aaaccaggcc 23340 catccctatc aacatggatc ccgtgtatcc ttctgggtaa gcactagaat tccaaggtct 23400 gcctggcatc ctcacctgtg ctggttccac gtcctgcagg aaattccgct gtcagaaatc 23460 ctcacggtgg agtccgccca gaacttcagc cttgtgccgc cgggcaccaa cccacactgc 23520 tttgagatcg tcactgccaa tgccacctac ttcgtgggcg agatgcctgg cgggactccg 23580 ggtgggccaa gtgggcaggg ggctgaggcc gcccggggct gggagacagc catccgccag 23640 gccctgatgc ccgtcatcct tcaggacgca cccagcgccc caggccacgc gccccacagt 23700 aagtcctccc acctcgggtc cttgagagaa tagatctaga tgggtggggc acggttctgg 23760 ggaatggaag ggccaaagag gaaagtgggc aatggtgggg ttgagaacgc agcttctgga 23820 ctcagcaggc ctgggttcaa actctgttaa tcactcctgt taatcccagc gctttgggaa 23880 gccaaggagg gaggatcact tgaggccagg agttcaagac cagcctgggc aacataatga 23940 gattccatct ctacaaaaaa taaaaacaat tagccaggtg tggtggtgca cacctgtagt 24000 tccaggtact tggaaggctg aggcaggaga attgcttgag cctgggagta gtgagtcatg 24060 attgcatcac tgcactccag tctgggtgac agagcaagac tctgtctcca aaacagaaaa 24120 aacaacaaca acaaaaatcc acaacaaatc tctgttaagc tcctggcctg atatgtggcc 24180 ctgggcatat cacttcccct ccatgagcct tgtcccaggt gctgataagt cctcatgcac 24240 ttactgagtg cctcctctgt gcgggacagt gctggggacc cagtggtggc caggacagcc 24300 caagacctgc cctcatgggg ctcagagtcc agtagggcag aatacccatc ttcagagagt 24360 gacagtccag ggtgggcagg gttgggacaa ggaagctagg gagctggagg agcccagagg 24420 ggtacctgac ccaatctggg tatatagggg ggcttcctgg aggaggtgac atctgaactg 24480 agatctggag gccgaggcag ggtgagatgt gggaaagaaa atgggaggtc attttaggca 24540 gaggcaaaaa atgttgagag agtaccaggt tcccaccctc tggagcttat aatccagtgt 24600 gggtgacaga cattgatcat taacccatac aagcaacgag tgtgatgcag agcatttgcg 24660 agagtaatcc aacttggtcc taggagtgac atttgagctt acacttgagg atgaggagga 24720 tttagctaag tctaggatga aggaaagagt attcctggca ggggaaacag catatgcaga 24780 gaccagaagg cagaagagag tttgctgtat ttgaggccga gcaaggaggc cagtgtgtca 24840 ggaatagcat gttgggggta gaagtcagag gtagatgagg gtctaggcca tggcttttag 24900 gccatttaag gggctcaggc ttcttcctga gggcactggg gagccatggc agagttgtga 24960 gcagaggagg gacagggtca gtcttgtgcc tcagtaagat ccctctggtt tctctgtggg 25020 aggtgagtag gaaggggcag gattggggca aggagaccag ggaaggggct gtggggtgag 25080 gacccagagt tggggggcga gcaggggcct agactggtgg aagagagaga cattcaaatg 25140 gcagaaggat cggactttag aaatgtctgg ctctggttgg gtttgtaggg ggaaaagttc 25200 aagggaagat gcaggagtca gtctgggctt tccctccaag actcagtttc cttctctgta 25260 caatggggtc agtctgcctc ccctggtgct gagatcctgg ggtaaaatgc tcagcaaaat 25320 catctgtaac atcactcctt tagccactca gcacatctca tttactcctc ctggtggctc 25380 tatgagggag gtccttttat tattcccatt ttctagatga ggaaactgag gttcgtagtg 25440 gacaagtcac cagcctgaag ttgcacattg tatcgaacat tggattcaaa tctgggtggc 25500 ctgactccca agtctgcttt tgcaggtatg ggtggagata atcctgagcc tggagtcccc 25560 tcacctctgt ctctcccctc tccctaggac aagcttctct gagcatctct gtgtccaaca 25620 gtcagatcca agagaatgtg gtgagacttc ctgcccccac ctgatgccct cccctcccac 25680 aaaccctcct cagctctctc gtctccttga ctcccccttc cccatttcca tttgcacccc 25740 tgacctgccc tgtcttcacc ctgtaggaca ttgccactgt ctaccagatc ttccctgacg 25800 aagtgctggg ctcagggcag tttggagtgg tctatggagg tgaggacact tcagagctaa 25860 cccagaggga gccccgggct gggggaagct gctgtggctc cagccctttc tttctggctc 25920 caacccttcc tttctgattg gtcacatgct cacctcccat gttgattggc ttagctagat 25980 cctgggtgga ctgattgcag gttctccttt tctcattggg aaaaaccaat ggacattcct 26040 cctgttatta ataggaaggg taaattcggc actctgattg gtcacagagg tagattttga 26100 ttggataggg aaggtagatt ctgcactctg attgaccaca gagctagaac ctagattctg 26160 attggataga gtagattctg cattcatatt ggccacagaa ctagttccta gattctgatt 26220 ggaaaagagg gtagattctg cactctggcc acagagctag atcctagatt ctgattgaat 26280 aggagggtag attctgcatt ctgattggcc acaggtctag atcctagatt ctgattggat 26340 tggagggtag attctgcatt ctgattggcc acaggctaaa tcctagattc tgattgtatg 26400 gggcgggtgg taaattttac actttgattt gccacagagc tagatcctag agttcaatag 26460 gacagggagg gtaacttcta cactctaaac tctaagactc agtttccttc tctgtataat 26520 agggtcagtc tgcctcccct ggtgctggtg tctctcccct gtccccagga ctcttatggg 26580 tcacacaaaa ctagatgcta gattccgact ggttataaat ccagtttccc atgttataca 26640 ttcccttctt cggagctttt tgtttgtttt ttgctttcct tctttctgcc tttactccca 26700 aggtgcacct caggtggcct tttcacgtat ctcctggggc cttccaactc tgcccaactc 26760 tggctgtctc catggtgggg ggcagaggtt ggcagaggtg gagatactcc tgccaggact 26820 gggtggtctt gctctctcat cccccatctc ttctactccc tgtgcaggaa aacaccggaa 26880 gacaggccgg gacgtggcag ttaaggtcat tgacaaactg cgcttcccta ccaagcagga 26940 gagccagctc cggaatgaag tggccattct gcaggtaacc accaggccgc cttccctttc 27000 tgcttcttcc tttcatgggc cagctgaccc agtgtagggg tggtcaggga aggcttcctg 27060 ggggagggca tgtgcatgtt gagactgaag gggagaaggt gttcttagca gagggaccag 27120 cctgtacaaa gacctggtga gagggagcat gaggttttct agaaaggagg tactgggaga 27180 tgaggccagg gaggagggcg gagccagacc ctttggactt tctcctgagg gtactggaga 27240 gccacagaag gcttttgagc aagggagggg caggatcagg tgtgtacgtt aggaaaatcc 27300 cgcaggctgc catctggagg gtgggtggaa agggaagtga ttgtagccag gaggctgagt 27360 ggggatctgg gtgggagaga ggggttaggc caggatagga ctggagaatg tgagaggggg 27420 tatggattta aaagatacag atgtgcagag ctctccccat ttctccaagc tccccctcct 27480 ccctcctgca accctgggcc tccaccagaa tttcaggatg taaagatcct tctgggccgg 27540 gcatggtggc tcacgcctgt aatcccagca ctttgggagg ctgaggtggg aggatcactt 27600 gaggccagaa gtttgagacc agcctggcca acatggcgaa accccatctc tatatttaaa 27660 tagaaagaaa aaaaagatcc ttctgggcac ctggcaggtg gggtggaggt gggcctgttc 27720 tgtcttggcc tgtgggaagc ccccttccct ctccaagtgc caatacccca gggacatcct 27780 tctccttgtt tgtcatcctc ctgctcctat acctgacccg ttggggtctg agtttgtggg 27840 ttacctgggc cctgaccccg ctccccaccc tgcagagcct gcggcatccc gggatcgtga 27900 acctggagtg catgttcgag acgcctgaga aagtgtttgt ggtgatggag aagctgcatg 27960 gggacatgtt ggagatgatc ctgtccagtg agaagggccg gctgcctgag cgcctcacca 28020 agttcctcat cacccaggtg cgtctgccct gcccgctgcc acccgcccct ccccatcagg 28080 tgtcagcttg gagaggccct gtatgcctag ggggtcaagc agacacttgg gggagtcaca 28140 atagcagata acagaaacca tcatcaggct gggcgcagtg gctcacaccc gtaatcccag 28200 cactttggga ggcccacgag gtcaggagat cgaaaccatc ctggctaaca tggtgaaacc 28260 ctgtctctac tagaaataca aaaaattagc cgggcatggt ggcaggcgcc tgtagtccca 28320 gctactcggg aggctgaggc aggagaatgg tgtgaacctg ggagatggag cttgcagtga 28380 gccgagatcg cgccactgca ctccagcccg ggcgacagag caagactcca tctcaagaaa 28440 aaaaaaaaaa aaaaaaagga accataatcg tacagaagta ataataacca taatagaaaa 28500 aataagccgg gcatggtagc acgtgtctgt ggtctcagct actcaggagg ctgaggcagg 28560 aggatcactt gatcccagga gttctgtgct gatcaggtgt cctcattaag tttggcatcc 28620 atgtggtgac ctcccaggag tgggggacca ccaggttgca aagcagccca ggttggaaat 28680 ggagcaggtc aaagctctct tactgatcag tagtgggatc acatctgtga agaggcattg 28740 cactccagcc tgggcaacat agcgagaccc cgcctctaaa aagaaagaaa gaaaaaagaa 28800 aaataatagt gacaataaca attaaaaata aagagtatgc caggcgcggt ggctcacgcc 28860 tgtaatccca acactttggg aggccaaggc gggtggatca cctgaggtca ggagtttgag 28920 accagcctgg ccaacatggt gaaaccctgt ctctactaaa aatacaaaaa ttagctgagc 28980 atggtggcag gcacctgtaa tcatagctac ttgggaggct gaggcaggag aatcccttga 29040 gcccaggagg cagaggttac agtgagctga gatcgtgcca ttgtactcca gcctggggga 29100 caagagtgaa acttcgtctc aaaaaaaaaa aaaataataa taataataat aaagagtaat 29160 cataataata gaaaaaaata gactagcggt aatgatagct atttttatta taaaaaataa 29220 atgatcagtc aggctccctg gacctgactt gactcatcta gaaaaaaggg gagtcaggca 29280 tggtggggta cacctgtaat cccagctact caggaagcta aggccagagg attgcttaag 29340 cccaggagtt tgagccagcc tgggcaacat agcaagagcc catctcaaaa acaggctggc 29400 tcatgcccgt aatcccagcg ctttgggagg ccaaggcaag aggatcgctt gaagccagga 29460 gttggagacc agcctaggcg acatagtgag atcccacctc tacaaaaagt aaaaaaaaaa 29520 atagaaaacc tagctggatg tggtgcctgg tagcacatgt ctgtagtcct agctgcttgg 29580 gaggaaggga gtggagaggc tctcttgaac ctaggtggtt gaggctgcag tgagctatga 29640 ccgtgccact gcactccagc ctgggtgaca gagcgagacc gtgtctcaaa accaaacaat 29700 agaaaaaacg ggcaagcagc cctttttctc tcattcattc attcagttgg tcaacaaaca 29760 ctccctagtc cctgctctgt gcttggtccc ttgctggtca gtgttgagga cacagggatg 29820 accaatacag ccccattctt agacagtgat agctcaggtg agcagggcta ggacaaggga 29880 ggctgataat ggtgatgata aataatgtgg tcactaacat ttattgagca cttactatgt 29940 gccaagcact cttcaaactc atttaatctt catagtaacc tgtgcagtag gtgctattat 30000 tatcaatccc cttttatggt tgaagaaact gagggtcaga gacatcaaat atcttgtcca 30060 gggtcacata gctggtggga tttgaaccta ggatctttgc ttttaactag tgatgtcaaa 30120 ctcatttgtg ttacattcaa acagattttc cttgtgtgcc tgtgtgcctg tgctttttgt 30180 ttgttttttt gagacagggt ctcgctctgt cacccgagct ggagtgcagt ggtacaatca 30240 tggctcactg cagccttgac ctcccgggtt caagcaattc tcctgcctca gcctcctgag 30300 tagctgagac aacaggcatc agccatcaca cccagctaat ttttataaag acatttttat 30360 aaagacttgc tatgttgccc aggctggtct tcaactcctg ggctcaagtg atcctcctga 30420 ctcggcctca gcctcgcaaa gttctgggat tacaggtgtg agccactgtg cccggcctct 30480 gttctgcgtt tctttttttt tggtggaggt gcacattaga ttcttatcac ttatattgtt 30540 caatggtttt atcccagtgt ttgcctcttt attttatatt tagtttttat ttaccatagg 30600 gttttattta ttttattttt tatttttttt tgagacggag tcttgctcta ttgcccaggc 30660 tggagtgcag tggcaccatc tcggctcact gcaagctccg cctcccaggt tcacaccatt 30720 ctcctacctc agcctcccaa gtagctggga ctacaggtgc ccaccaccac gcccggctaa 30780 ttttttgtat tttcagtaga gacagggtct cactgtgtta accaggatgg tctcgatctc 30840 ctgacctcgt gatccacccg cctcggcctc ccaaagtgct gggattacag gtgtgagcca 30900 ccgcgcctgg cctattttat tttttttttt gagacagggt ctcattttgt cacccaggct 30960 ggagtgcagt ggtgtaatca tagttcactg cagcctcaaa ctcctaggct gaagcaattc 31020 tcctatctca gcctcctgag ttaactggaa ccacaggcat gagccaccac gtccagctaa 31080 tttttttttt tttttttttt aatgtttttg tagagacaag gtctcgccat gttgcccagg 31140 ctggtcttga actcctgggc tcgagcgatc ctcccatctc agtctcctga gttagctgga 31200 accacaggca tgagccatta cacctggcta attttttttt atgtttttgt agagacaggg 31260 tcttgccatg ttgggtctcg aactcctggg cttaagtggt cctcttgctg cagcctccca 31320 aagttctggg ttacaggcat gagccactgc gtccagccgg ccatagagtg gaacttttac 31380 gatgttaaat atccccttgt gtggtttctg tgtttcacat ccttcctaga aaggcttcct 31440 tctggtgggt gccttgcctt cttctgagac atctctgtgg gtctcagagc catcgttgct 31500 gtgttccctt taccctggcc cagcaccctt atcctctcag gcagtgtgcc tgtgtttgtc 31560 aggctggctt atggggtggg gacagaaacc cactgatgca ccctcatcca gactttatta 31620 tttatgtatt tttgagacag agtctcgctt tgttgcccag gctggagcgc agtgacacga 31680 tctcggctca ctgcaccctc tgccccctgg gttcaggtga ttctcctacc tcagcctccc 31740 gagtagctgg gattataggt gtgtgccacc atgcctggct aatttttgta attttagtag 31800 agatggggtt tcatcatgtt gcccaggcca gtctcaaact cctgacctca agtcatctgc 31860 ctgcctcagc ctcctgaagt gctgggatta caggcatgag ccatcgtgcc cggccacatc 31920 cagacttcag gtgtggaaag gaatcatggt tctcacaggt ggctgctttc agcagctgag 31980 ggggtttctc tttctggcct tcatctcttc ctctcttttt gcctgctcgc tcttctttct 32040 ctctctctct ctctctgcag atttctgctt tctgggctct tgcctgcccc acacctaagc 32100 cctgtgctaa gccctttacc tcctgagctt atgtaggcct caccaccatc ctaggaggta 32160 ggtattgtta taaaccccat tttatagatg aggaaactga ggctcaggga gttagcagtc 32220 tccctcgagg tcacagccaa gtagctttcc agccaagatt tgagtctgga tctatctagc 32280 ttccaacctg ccctctttct tttctttttt tttttttttt tttgagacga agtctcactc 32340 tgtcacccag gctggagtgc aatagtacag tctcagctca ctgcaacctc tgcctcccag 32400 gttcaaacaa ttgtcccacc tcagcctcct gagtagctgg gactacaggt gcgtcccagt 32460 acaccgggct aatttttgta tttttagtag agacggggtt tcactatgtt ggccaggcta 32520 gtcttgaact tctgacctcg tgatccaccc gcctcagcct cccaaaatgc tgggattaca 32580 ggcgtgagcc accatatccg gccaatgttt tttttttttg gagatggagt ctcgctctgt 32640 tgcccaggct ggagtgcagt ggcgctatct cagctcactg caacctctgc ctcccaggtt 32700 caaatgattc tcctgcctca gcctcctgag tagctgggaa cacaggcaca cgccaccatt 32760 cctggctgat ttttgtattt ttagtagaga tggggtttca ccatgtcgat caggctggtc 32820 ttgaactttt gatctcgtga tctgcccgcc tcagcctccc aaagtgctgg ggattacagg 32880 cgtaagccac cgtgcccggc ctaacctgcc ctctttgttc acatgaactg ggagaaaatc 32940 aactgacaaa atctggaaat gggcggggcg aggtggctca cgcctgtcat cctagaactt 33000 tgggaggcca aggcagatgg atcacctgag gtcaggagtt ttgagaccag cctggccaac 33060 atggtgaaat cccatcttta ctaataatac aaaaattagc caggtgtggt ggcattcacc 33120 tgtaatccca gctactgggg aggctgaggc acaagaattg cttgaacctg ggaggtggaa 33180 tttgtggtga gtcgaggtca tgccgttgca ctccagcgtg ggcaacagag tgagactcca 33240 tctcaaaaaa acaatctgga gatgacatat acaacacatg catctttcca gcttggtctc 33300 ccagtctgta gaatgaggag gttggtcagg catggtgggt cgtgcctatt atctcaaggt 33360 ttgggtagct gaggtgggaa gatcatttga ggccaggagt tttagaccag cctgggcaac 33420 atagcgagat gccatctcta caaaaagatt tttttaaaaa agaaaacaat cagaataaac 33480 acaagtattt aaactctgag acagatacac aagtatttaa actccgagac agataataat 33540 tgcagttgta caacactcta tgcttctggt gtacttggca ttttgagtta cagagaatca 33600 agaaatatga ttctcacaga tgaatggtta caaatggtaa tttttttttt aatcagctca 33660 ccttatcata ggaacagata cagcaggaga agctttattt aagagacaca aacaaatata 33720 tttaccaaca agccatcaca aaaataataa ctaataacaa caacagtaac agctaacata 33780 cagtggttag ctatcctaag cgttttacat gcatctttag atatgcttta aaccttatag 33840 caacctgtaa ggttggtact cttttttttt ctgagaggca tctcactctg tcgcccaggc 33900 tggaagtgca atggcgcgat gtcgactcac tgcaacctcc acctctccag ttcaagcgat 33960 tttcctgcct cagcctcccg agtagctggg actacaggcg cccaccacca cgcctaattt 34020 ttgtattttt aatagaggca gggttttgct atgttggcca ggatggtgtc taactcctga 34080 cctcaggtga tccacctgcc tcagccttcc aaagtgctga gattacaggc atgagtcacc 34140 atgcccagcc aaagtttttt gtaaggatga aaaatatttt ttttaaaaat gaaatcaggc 34200 tgggcacagt ggctcacgcc tataatccca gcactttggg aggccaaggt tggtggatca 34260 cgaggtcagg agttcaagac cagcctgacc aacatgatga aaccccgtct ctactaaaaa 34320 tacaaaaatt agccgggcat ggtggtgtgt gcctgtaatc ccagctgctc aggaggctga 34380 ggcaggagaa tcaggaggcc ttctcaaaaa aaaaaaaaaa aaggaatcaa agcccgacat 34440 ggtggtggtg gcacatgcct gtagtcctag ctatttggga gactgaggct ggaggatcac 34500 ttaaccccag gagtttgagg ctgtagaatg atactgcact tcagcctggg tgacagaggg 34560 agactccatc tcttcaaaaa aaaaatgggt gaggtggggg tggctcacgc ctgttatcca 34620 agcactttgg gaggctgagg tgggtggatc acttgagtgc aggagtttga gaccagcctg 34680 ggcaacatgg tgagacactg tctctacaaa tacaaaaatt agtcaggtgt gatggtgtgt 34740 gcctataatc ccagttacta gggaggttga ggtgggagga tggatttagc ctgggaggtc 34800 gaggtgcagt gagctgtgat cccgcctctg tgctctggcc tgagtgacag agcaagactc 34860 tgtctcaaaa aaaaaaaaaa aaaaaataga atcacatagt tggatcttgg aaatgcctgc 34920 tctgtgagta gcattcagga gtttaccaca tgctagaaga tcttgggatc ttacagcccc 34980 actcatctag cccagacttt ctagtttaca tttaactctt atctctcaga tgtaaatggt 35040 tctatgattc tgagattctt tggtgctcca gtgcctcctg tttccctggc tggggtgtct 35100 gcaggggtgt gtaggaaggc atggatgggg ccaggcgcag tggctcactc acgcctgtaa 35160 tcccagcatt ttgggaggcc aaggtgggtg gatcacttga gtccaggagt ttgagaccag 35220 cctggtcaac atggtgaaac cctgtctcta ctaaaaataa aagaaaaaat tatcagagca 35280 agtctgggcc cggtggctca cgcctgtaat cccagcactt tgggaggccg aggtggggga 35340 atcacgaggt caggagtttg agaccagcct ggccaacatg gtgaaacccc atctctacta 35400 aaaatagaaa aaattagctg ggcatagtgg ccagcgcctg taatcccagc tactcgggag 35460 gctgaggcag gagactcact tgagccctgg aggtagaggt tgcagtgagc cgagatcgtg 35520 ccactgcact ccagcccagg cgacagagtg agactccgcc tcaaaaagaa aaaaaaaaat 35580 tagctgggca tggtggtgca cgcctgtagt cccagctact tgggaggctg aggcaggaga 35640 atcacttgaa cccaggaggt aggggttgca gtgagctgag atcatgccac tgcacttcca 35700 gcctgggcta cagagcgaga ctctgtctca aaaaaaaaaa aaaaaaagta tggatgggtt 35760 tggagggctg gctgctgagg ttgggatttg gctgagtacc tatctacctt tccttactgg 35820 gcccatctgc tcccctcaga tcctggtggc tttgagacac cttcacttca agaacattgt 35880 ccactgtgac ttgaaaccag aaaacgtgtt gctggcatca gcagacccat ttcctcaggt 35940 cagttatgtc ccctcctgat ttggggaaat ccaggcaaca ctgatggccg gggtgggggt 36000 ggggaagggg attatactaa tcaagatgtg ggggcgaggc acagtggctc ttgcctgtaa 36060 tcagcatttt gagaggctga ggcaggagga tcatttgagc ccaagagttt gagaccagcc 36120 tgggcaacat agcgagacct catctataca aaaaatgaaa aaaaaaatag ccgggaatgg 36180 tggcgtgcgc ctatagtcct agctgcttag gaggctgaga tgggaggatt gcttgagccc 36240 aggagttggt ggctgcagtg agctatgatt gtgccactgc actccagcct gaataacaga 36300 gtgagagctg tctcttaaaa aaaaaaaaaa aagactgggt gcggtggctc acgcctgtaa 36360 tcccagcact ttgggaggcc gaggcgggca gttcacgagg tcaggagatc gagaccatcc 36420 tggctaacac ggtgaaaccc cttctctact aaaaatacaa aaaaaaatta gcggggcgtg 36480 gtggtgtgtg cctgtagtcc cagctacttg ggaggctgag ttaggagaat ggcatgaacc 36540 cgggaggcgg agcttgcagt tagccgagat cacgccactg cactccagcc tgggtgacag 36600 agcgagagag cgagactctg tctcaaaaaa aaaaaaaaaa atatatatat atatatatat 36660 atagtttatc ccaacatata gcactttatt caacatgtag tcaacataaa aattattaag 36720 gccaggggag gtggctcatg cctataatcc ccgcactttg ggaggccaag atgggaagac 36780 ggcttgagac caggagttca agtctgaagt gagctatgat tgtgccactg cactccagct 36840 ggggtgacag agcaagaccc tgtctcttaa aaaagaaaca aaactcaatg aaacattctg 36900 cttgtttttc atactatgtc ttcaaaatct ggtgtgtata acagttgggg aaatagattg 36960 acatgcccaa gttgttccaa acatatttaa aagttttctg gttgggcgca gcggctcatg 37020 cctataatcc cagcactttg ggaggctgag gcgggcagat cacttgaggt ctggagttgg 37080 ataccagtct ggctaacatg gcgaaacccc gtctctacta aaaatacaaa aattagctgg 37140 gcatggtggc gggaacctgt aatcccaggt tctcaggagg ctgaagcagg agaattgctt 37200 gaacccagga gggtggaggt tgcggtgagc cgagatcaca ccactgcact ccagcctgga 37260 cgacagacca agactcgtct caaaaaaata ataataaaat aaaaatttta aaaaagatcc 37320 ataggaaagt atagatcttg gaaaagagaa agagctataa gatctgtaga aagggcagag 37380 tacctcagga aagggtggct gtcacattga gattcaggtc aggggttgag gcgtggctgg 37440 tttcaaaggt gacagaggct tcaggcttca aggatttggg gctctatcct gcaagcaaca 37500 gtgagccaag gaagggtttt gaacagggaa aggacagtac atgaacagag ctgggaacca 37560 aggctgagag gtaggcagca gagcaagacc ttgaacccag gtcttgctgg ctccaaagcc 37620 tgtccatgac cttagactgc agccattaac aatgagggta tggggccagg tgtggtgtct 37680 catgcctgta atcccagcac tttgggaggc tgaggcagga ggaacacctg aggtcaggag 37740 tttgggacca gcctggctga tgtggtgaaa tgtcgtctct actaaaaata caaaaattag 37800 ccaggcatgg tggcgggtcc ctgtgatccc agctattcgg gaggctgagg caggagaatt 37860 gcttgaaccc gggaggcaga ggttgcagtg agccaagatc acgctactgc actccagcct 37920 gggcgacaga gcgagactcc gtctcaaaaa aaataaaaca atgaaggaaa ggtaggcata 37980 caccatactg tctgccagct accgcagtca gcacccactc ctacctaatc cccaggaaag 38040 cctgagagga ggctgctatc aacaaccccc caatacagat gacaaaatca aggcctggag 38100 aaattaggtc cttgacctga gatcatcgag ggtcattctg tgctagacac tgctcctaac 38160 acgttgcata catttctctt tcagtctaaa caagcaccct ttaaggtagg gactgttaag 38220 atctccatta tgtttcatgt tttttttgtt tgttttttga gacggagtct cgctgtgtca 38280 cccaggctgg aatgcagtgg tgcgatctcg gctcactgca acctctgcct cccaggttca 38340 ggcgattctc ctgcctcagc ctcctgtagt agctgggacc gcaggcgtgt gctaattttt 38400 gtatttttag tagagatggg gtttcatcgt gttggccagg ctggtctcga actcctgacc 38460 tcaaatgatc catcttcctt ggcctcccaa agtgctgaga ttgcaggcat gagccaccac 38520 gccccaatca tgtatatttt gaggctatta aaaaaaatct gcattattca aaagaggaaa 38580 cagcgaccca ttggaggtgg cagaggtata gcagcagcta gcatttattg tgcaccaact 38640 gaatgccaaa tattgtcctg tgggctttgg atggtttaat tcactaacca tcatggcagt 38700 cctctgagat aggtgctctt ctgctcttct tcctatagat ggggaaactg aggcacagag 38760 gggggaagtc acctgcccag ggttgctcag ctagtgagcc aaggagcctg gattcaaacc 38820 agcatccagc tttctctgga ataccatgga gggtggtgtg gtggggatgc tggggtgggt 38880 gcggctccat cacctggtgg agcctccatc ccttgccctc tgcaggtgaa gctgtgtgac 38940 tttggctttg ctcgcatcat cggcgagaag tcgttccgcc gctcagtggt gggcacgccg 39000 gcctacctgg cacccgaggt gctgctcaac cagggctaca accgctcgct ggacatgtgg 39060 tcagtgggcg tgatcatgta cgtcagcctc agcggcacct tccctttcaa cgaggatgag 39120 gacatcaatg accagatcca gaacgccgcc ttcatgtacc cggccagccc ctggagccac 39180 atctcagctg gaggtgcctg gggcccgcct accccatggg cgggtgggtt gtggggtggg 39240 gctggagaag tgggcggagc catgagaggg gggtggaccc ggaaacagcc tggcaccttg 39300 ggggtggagc ccagtgctgg ggcgggccta ctggagggat gtggctacag gaggagccgt 39360 cctgtaaaag atgggctggg actcaggcct agactaggtt acttgggctg gaaaccaagt 39420 gccccagaag cgctgaggac acttggaacc ttaggggggc tgagtgagac ttggcttgtc 39480 tagggtggga ccaggaaagg gactggactt gagggtacca aagggctgcg gtgaccagga 39540 gaaggggctg agcctcccaa ggcattggct gggacctgga gcctttgggt ttacgacccc 39600 aaaagggtca gccttgcaaa aaggaggcac cggtgggtag ggttgagaaa caagggcatg 39660 gctactttgc tgtgtactgg ggccgtgact tgggtgaaga tgggcctgaa gcctggggtc 39720 ggttcagtga ccaagggagc cagtctaggg acgtggccgt ggagggtttc cgaagaggtc 39780 caggaacagg gctgaccctg agtcctggaa gctgggagtg gatgggagtg gggaggagaa 39840 gggagccagg actgaggcag acattgcact ctgcattctg gggctttggt gttgtggctg 39900 ggcctgatga agtggcaccg ggcctggtga cttgaaccta cttgggaatg ggtctgtaac 39960 tttccctgct tggaaaagtt aagtcctaag gcctggagct ttgaggctgg gtgtgggatg 40020 gcatgtttag agggccagag gcagggctaa gatactgggg tgtgtcagaa gccaggagaa 40080 caagggacct gtgttggagc cagggagctc aggaagacag atggagtatg ggaagggggg 40140 ggatcattca ttcatttatt tataaccatt tattcaacaa gtacattcat gtatttgtaa 40200 ccattgattc aacatgttga gtgcccacga tgtgccaggc attgactgtt ccagctctgg 40260 gaatactgtg atgacttgga cagaaggggt caggtgcagg gtagctcatt gagtggtccg 40320 cgaagggtgg aaaggggaag ggtcctctct ggagggtgcg gcttcatgga gcaggtggag 40380 cagggtgaca cggaggttgc tcggtgcagg acaagacaag gtcttggtgg tggtctaaga 40440 gcatgggccc taagcagtga gaatgtggat tgacttgagt cctggagtaa tattgggggt 40500 gctcaacact ggcttttttt tttttttttg aggtggggtc tcgctctttc acccacgctg 40560 gagtgcagtg gcgtgatctc ggctcactgc aacctccacc tcttgggttc aagggattct 40620 cctgcctcag cctcccgagt aactgggatt acaggcacac agcaccatgc ctggctcatg 40680 ttttatattt ttagtagaga cagggtttcg ccatgttagc caggctggtc ttgaactcct 40740 gacctcaagt tatttgcccg cttcagcctc ccaaagtgct gggattgcag gcataagcca 40800 tcacaccccg ccagcattgt cttttgagac ccactcagaa gtccctcagt aaaagtgcat 40860 cgagtgtgca caagtgaatt taagtgtggt tgcacctgtg tgaggatcac agaatcctgt 40920 gggtgttgac gggagcaggg tgcctgtgtg caccaggcct ctcctcggat gggttcatac 40980 agtgaagcct tgtccttcat ggcttcccat caaggagaga gcctcggatg agtgctggct 41040 tgtcttgaag cttgacattc gctagtcctc tttttcacaa tgaacaggcc tatctctgag 41100 ccttctgcag gcaatggtga ctaactacca tctgatgaca ttttgttttg ttttgttttg 41160 ttttgagacg gagtttcgct tttgtcaccc gggctggagt gcagtggcac gatcttggct 41220 cactgcaacc tctgcctcct gagttcaagc gattctgcct cagcctcctg agtagctggg 41280 actacaggca tgcgctacca tgcccagcta attttttgta tttttagtag agacggggtt 41340 tccgtgttgg ccaggcttgt ctcgaactcc tgacctcggg tgatccaccc gcctcggcct 41400 cccaaagtgt tgggattaca ggcatgagcc accgcgccca gcctgatgac atagatgctc 41460 cctgatttgc actggggtta gataaacctg ataaacccat tgcccattgt aaattgaaaa 41520 tatcataagt tggtcaggcg cagtggctga agcccataat cccagcacct tgggaggcca 41580 aggtaggcag attgcttgag cccaggagtt caagaccagc ctgggcaatg tatctctaca 41640 aaaaatacaa aaattagccg gccatagtga caggtgcttg tagtcccagc tggctgctca 41700 ggaggctaag gcaggagaat caattaagct ggggaggtgg aggcttcagt gagcattgat 41760 cacgccactg cacttcagct tgggtaacaa tgagaccctg tctcaaaaaa aaaaaaggaa 41820 gtattgtagg ttgaaaatcc atttaggccg ggcgcagtgg ctcatgcctg taatcccaac 41880 aatttgggag gccaaggcag gcggattgct tgaggtcagg agttagagac cagcctggcc 41940 aatatggtga aaccccatct ctactaaaaa tacaaaaagt tagcaggaca tggtgacaca 42000 cacctgtatt cctagctact tgggaggctg aggcaggaga atcacatgaa cccgggaggc 42060 ggaggttgca gtgagccaag atcgtgccat tgcactccag cctgggcgac agagcgagac 42120 tctgtctcaa taaataaata agtaaaaata aaaagaatag tacaggtgta attgtatgta 42180 cctgtatatg acaaaaagaa aaaaaaaggt gacatagggg aatggggaaa ttgaagtaga 42240 gaacaggtga agagagggag ctggtgtgaa catgcatggg caggaggaga caaatttgta 42300 atgtaatgag gaaatgggtg ggtgagtgat tggcacaggt gaggcttctg agccacctga 42360 gctggtgcag aaggaaggtg ttgatggcag gcaggtaggc tagggggtgc ctattggagg 42420 aggagtgacc cttgacctgt agggcttgac ctgtttctct ttcctgtgca gccattgacc 42480 tcatcaacaa cctgctgcag gtgaagatgc gcaaacgcta cagcgtggac aaatctctca 42540 gccacccctg gttacaggtg atgcaggggg cagggctggc ccattggctg gattggagga 42600 aggggtggga gtagatcgct tattggctag gcaggttgtg aaggatgtag gtttccttgg 42660 gtctggaatg tggctaggcc tcccattggc tgggtgcagg aagagggggt ggagctaaat 42720 gtctactggc tgggtgggtt gcagagggta tggcttcacc ttcattggta cccagctctc 42780 agtggcaaac cagaggatat ccaggcactg ctccaatgca gaccccaagc taaccccagt 42840 tctctcgggc ccaggagtac cagacgtggc tggacctccg agagctggag gggaagatgg 42900 gagagcgata catcacgcat gagagtgacg acgcgcgctg ggagcagttt gcagcagagc 42960 atccgctgcc tgggtctggg ctgcccacgg acagggatct cggtggggcc tgtccaccac 43020 aggaccacga catgcagggg ctggcggagc gcatcagtgt tctctgaggt cctgtgccct 43080 cgtccagctg ctgccctcca cagcggttct tcacaggatc ccagcaatga actgttctag 43140 ggaaagtggc ttcctgccca aactggatgg gacacgtggg gagtggggtg gggggagcta 43200 tttccaaggc ccctccctgt ttccccagca attaaaacgg actcatctct ggccccatgg 43260 ccttgatctc agcacacggc actctcgaat cattactctg ttgtaccaac atggagttca 43320 tctggaagga ggactgcctg aaaagaggaa ggatggaagg ggtggggaga gaggactgat 43380 gggagaggag tcttggaagg aggacgagct ggggtagaaa atatacagga agagtgccag 43440 gagagaagat gagaagggag agggaggagt aatggaggag gagttggaaa ctggggagag 43500 atggaaggaa tgtgactgga gggtagagaa cttggagaaa aagtaatctc atggtttgtg 43560 atgactgatt ttttatttgg tggtggtgtt actactaatc acaactatta attcaggctg 43620 ggtgtggtgg ctcatgccta taatcccagc aatttgggag gccgaggcag gcagatccct 43680 tagatctcag gagtttgaga gcagcctggc caacgtggtg aaactccctt tctacaaaaa 43740 gttcaaaaat tagccaagtg tggtggcttg cacctgtggt cccagctact tggaggttga 43800 ggctagagga tcgcttgagc ccaggaagca gagattgcag tgagccaaga tcacacacca 43860 ctgcactcta gcctgggcaa gagagtgaga ccctgtctca aaagtcaaat aataaaatgc 43920 agttagccca agtctgatcc atactagaaa 43950 4 894 PRT Homo sapiens 4 Ala Ala Ala Ala Ala Ala Ala Ala Leu Val Pro Gly Ser Gly Pro Gly 1 5 10 15 Pro Ala Pro Phe Leu Ala Pro Val Ala Ala Pro Val Gly Gly Ile Ser 20 25 30 Phe His Leu Gln Ile Gly Leu Ser Arg Glu Pro Val Leu Leu Leu Gln 35 40 45 Asp Ser Ser Gly Asp Tyr Ser Leu Ala His Val Arg Glu Met Ala Cys 50 55 60 Ser Ile Val Asp Gln Lys Phe Pro Glu Cys Gly Phe Tyr Gly Met Tyr 65 70 75 80 Asp Lys Ile Leu Leu Phe Arg His Asp Pro Thr Ser Glu Asn Ile Leu 85 90 95 Gln Leu Val Lys Ala Ala Ser Asp Ile Gln Glu Gly Asp Leu Ile Glu 100 105 110 Val Val Leu Ser Arg Ser Ala Thr Phe Glu Asp Phe Gln Ile Arg Pro 115 120 125 His Ala Leu Phe Val His Ser Tyr Arg Ala Pro Ala Phe Cys Asp His 130 135 140 Cys Gly Glu Met Leu Trp Gly Leu Val Arg Gln Gly Leu Lys Cys Glu 145 150 155 160 Gly Cys Gly Leu Asn Tyr His Lys Arg Cys Ala Phe Lys Ile Pro Asn 165 170 175 Asn Cys Ser Gly Val Arg Arg Arg Arg Leu Ser Asn Val Ser Leu Thr 180 185 190 Gly Val Ser Thr Ile Arg Thr Ser Ser Ala Glu Leu Ser Thr Ser Ala 195 200 205 Pro Asp Glu Pro Leu Leu Gln Lys Ser Pro Ser Glu Ser Phe Ile Gly 210 215 220 Arg Glu Lys Arg Ser Asn Ser Gln Ser Tyr Ile Gly Arg Pro Ile His 225 230 235 240 Leu Asp Lys Ile Leu Met Ser Lys Val Lys Val Pro His Thr Phe Val 245 250 255 Ile His Ser Tyr Thr Arg Pro Thr Val Cys Gln Tyr Cys Lys Lys Leu 260 265 270 Leu Lys Gly Leu Phe Arg Gln Gly Leu Gln Cys Lys Asp Cys Arg Phe 275 280 285 Asn Cys His Lys Arg Cys Ala Pro Lys Val Pro Asn Asn Cys Leu Gly 290 295 300 Glu Val Thr Ile Asn Gly Asp Leu Leu Ser Pro Gly Ala Glu Ser Asp 305 310 315 320 Val Val Met Glu Glu Gly Ser Asp Asp Asn Asp Ser Glu Arg Asn Ser 325 330 335 Gly Leu Met Asp Asp Met Glu Glu Ala Met Val Gln Asp Ala Glu Met 340 345 350 Ala Met Ala Glu Cys Gln Asn Asp Ser Gly Glu Met Gln Asp Pro Asp 355 360 365 Pro Asp His Glu Asp Ala Asn Arg Thr Ile Ser Pro Ser Thr Ser Asn 370 375 380 Asn Ile Pro Leu Met Arg Val Val Gln Ser Val Lys His Thr Lys Arg 385 390 395 400 Lys Ser Ser Thr Val Met Lys Glu Gly Trp Met Val His Tyr Thr Ser 405 410 415 Lys Asp Thr Leu Arg Lys Arg His Tyr Trp Arg Leu Asp Ser Lys Cys 420 425 430 Ile Thr Leu Phe Gln Asn Asp Thr Gly Ser Arg Tyr Tyr Lys Glu Ile 435 440 445 Pro Leu Ser Glu Ile Leu Ser Leu Glu Pro Val Lys Thr Ser Ala Leu 450 455 460 Ile Pro Asn Gly Ala Asn Pro His Cys Phe Glu Ile Thr Thr Ala Asn 465 470 475 480 Val Val Tyr Tyr Val Gly Glu Asn Val Val Asn Pro Ser Ser Pro Ser 485 490 495 Pro Asn Asn Ser Val Leu Thr Ser Gly Val Gly Ala Asp Val Ala Arg 500 505 510 Met Trp Glu Ile Ala Ile Gln His Ala Leu Met Pro Val Ile Pro Lys 515 520 525 Gly Ser Ser Val Gly Thr Gly Thr Asn Leu His Arg Asp Ile Ser Val 530 535 540 Ser Ile Ser Val Ser Asn Cys Gln Ile Gln Glu Asn Val Asp Ile Ser 545 550 555 560 Thr Val Tyr Gln Ile Phe Pro Asp Glu Val Leu Gly Ser Gly Gln Phe 565 570 575 Gly Ile Val Tyr Gly Gly Lys His Arg Lys Thr Gly Arg Asp Val Ala 580 585 590 Ile Lys Ile Ile Asp Lys Leu Arg Phe Pro Thr Lys Gln Glu Ser Gln 595 600 605 Leu Arg Asn Glu Val Ala Ile Leu Gln Asn Leu His His Pro Gly Val 610 615 620 Val Asn Leu Glu Cys Met Phe Glu Thr Pro Glu Arg Val Phe Val Val 625 630 635 640 Met Glu Lys Leu His Gly Asp Met Leu Glu Met Ile Leu Ser Ser Glu 645 650 655 Lys Gly Arg Leu Pro Glu His Ile Thr Lys Phe Leu Ile Thr Gln Ile 660 665 670 Leu Val Ala Leu Arg His Leu His Phe Lys Asn Ile Val His Cys Asp 675 680 685 Leu Lys Pro Glu Asn Val Leu Leu Ala Ser Ala Asp Pro Phe Pro Gln 690 695 700 Val Lys Leu Cys Asp Phe Gly Phe Ala Arg Ile Ile Gly Glu Lys Ser 705 710 715 720 Phe Arg Arg Ser Val Val Gly Thr Pro Ala Tyr Leu Ala Pro Glu Val 725 730 735 Leu Arg Asn Lys Gly Tyr Asn Arg Ser Leu Asp Met Trp Ser Val Gly 740 745 750 Val Ile Ile Tyr Val Ser Leu Ser Gly Thr Phe Pro Phe Asn Glu Asp 755 760 765 Glu Asp Ile His Asp Gln Ile Gln Asn Ala Ala Phe Met Tyr Pro Pro 770 775 780 Asn Pro Trp Lys Glu Ile Ser His Glu Ala Ile Asp Leu Ile Asn Asn 785 790 795 800 Leu Leu Gln Val Lys Met Arg Lys Arg Tyr Ser Val Asp Lys Thr Leu 805 810 815 Ser His Pro Trp Leu Gln Asp Tyr Gln Thr Trp Leu Asp Leu Arg Glu 820 825 830 Leu Glu Cys Lys Ile Gly Glu Arg Tyr Ile Thr His Glu Ser Asp Asp 835 840 845 Leu Arg Trp Glu Lys Tyr Ala Gly Glu Gln Arg Leu Gln Tyr Pro Thr 850 855 860 His Leu Ile Asn Pro Ser Ala Ser His Ser Asp Thr Pro Glu Thr Glu 865 870 875 880 Glu Thr Glu Met Lys Ala Leu Gly Glu Arg Val Ser Ile Leu 885 890 

That which is claimed is:
 1. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a protein comprising the amino acid sequence of SEQ ID NO:2; (b) a nucleotide sequence consisting of SEQ ID NO:1; (c) a nucleotide sequence consisting of SEQ ID NO:3; and (d) a nucleotide sequence that is completely complementary to a nucleotide sequence of (a)-(c).
 2. A nucleic acid vector comprising a nucleic acid molecule of claim
 1. 3. A host cell containing the vector of claim
 2. 4. A process for producing a polypeptide comprising culturing the host cell of claim 3 under conditions sufficient for the production of said polypeptide, and recovering said polypeptide from the host cell culture.
 5. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:1.
 6. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:3.
 7. A vector according to claim 2, wherein said vector is selected from the group consisting of a plasmid, virus, and bacteriophage.
 8. A vector according to claim 2, wherein said isolated nucleic acid molecule is inserted into said vector in proper orientation and correct reading frame such that the protein of SEQ ID NO:2 may be expressed by a cell transformed with said vector.
 9. A vector according to claim 8, wherein said isolated nucleic acid molecule is operatively linked to a promoter sequence. 